Pluripotent stem cell-derived oliogodendrocyte progenitor cells for the treatment of spinal cord injury

ABSTRACT

The present disclosure provides methods and compositions for making and using pluripotent stem cell-derived oligodendrocyte progenitor cells.

PRIORITY

This application is a Divisional application of U.S. Nonprovisionalapplication Ser. No. 16/409,760, filed on May 10, 2019, which is aContinuation application of U.S. Nonprovisional application Ser. No.15/156,316, filed on May 16, 2016, which issued as U.S. Pat. No.10,286,009 and claims priority to U.S. Provisional Patent ApplicationNo. 62/162,739, filed on May 16, 2015, the entire contents of which arehereby incorporated by reference.

FIELD

The present disclosure relates to the field of stem cell biology andoligodendrocyte progenitor cells. More specifically, the presentdisclosure relates to oligodendrocyte progenitor cell compositions andmethods of using the same.

BACKGROUND

Over 12,000 Americans suffer a spinal cord injury (SCI) each year, andapproximately 1.3 million people in the United States are estimated tobe living with a spinal cord injury. Traumatic SCI most commonly impactsindividuals in their 20s and 30s, resulting in a high-level of permanentdisability in young and previously healthy individuals. Individuals withSCI not only have impaired limb function, but suffer from impaired boweland bladder function, reduced sensation, spasticity, autonomicdysreflexia, thromboses, sexual dysfunction, increased infections,decubitus ulcers and chronic pain, which can each significantly impactquality of life, and can even be life threatening in some instances. Thelife expectancy of an individual suffering a cervical spinal cord injuryat age 20 is 20-25 years lower than that of a similarly aged individualwith no SCI (NSCISC Spinal Cord Injury Facts and FIGS. 2013).

The clinical effects of spinal cord injury vary with the site and extentof damage. The neural systems that may be permanently disrupted belowthe level of the injury not only involve loss of control of limb musclesand the protective roles of temperature and pain sensation, but impactthe cardiovascular system, breathing, sweating, bowel control, bladdercontrol, and sexual function (Anderson K D, Fridén J, Lieber R L.Acceptable benefits and risks associated with surgically improving armfunction in individuals living with cervical spinal cord injury. SpinalCord. 2009 April; 47(4):334-8.) These losses lead to a succession ofsecondary problems, such as pressure sores and urinary tract infectionsthat, until modern medicine, were rapidly fatal. Spinal cord injuryoften removes those unconscious control mechanisms that maintain theappropriate level of excitability in neural circuitry of the SpinalCord. As a result, spinal motoneurons can become spontaneouslyhyperactive, producing debilitating stiffness and uncontrolled musclespasms or spasticity. This hyperactivity can also cause sensory systemsto produce chronic neurogenic pain and paresthesias, unpleasantsensations including numbness, tingling, aches, and burning. In recentpolls of spinal cord injury patients, recovery of ambulatory functionwas not the highest ranked function that these patients desired toregain, but in many cases, relief from the spontaneous hyperactivitysequelae was paramount (Anderson K D, Fridén J, Lieber R L. Acceptablebenefits and risks associated with surgically improving arm function inindividuals living with cervical spinal cord injury. Spinal Cord. 2009April; 47(4):334-38).

There are multiple pathologies observed in the injured spinal cord dueto the injury itself and subsequent secondary effects due to edema,hemorrhage and inflammation (Kakulas B A. The applied neuropathology ofhuman spinal cord injury. Spinal Cord. 1999 February; 37(2):79-88).These pathologies include the severing of axons, demyelination,parenchymal cavitation and the production of ectopic tissue such asfibrous scar tissue, gliosis, and dystrophic calcification (Anderson DK, Hall E D. Pathophysiology of spinal cord trauma. Ann Emerg Med. 1993June; 22(6):987-92; Norenberg M D, Smith J, Marcillo A. The pathology ofhuman spinal cord injury: defining the problems. J. Neurotrauma. 2004April; 21(4):429-40). Oligodendrocytes, which provide both neurotrophicfactor and myelination support for axons are susceptible to cell deathfollowing SCI and therefore are an important therapeutic target (AlmadA, Sahinkaya F R, Mctigue D M. Oligodendrocyte fate after spinal cordinjury. Neurotherapics 2011 8(2): 262-73). Replacement of theoligodendrocyte population could both support the remaining and damagedaxons and also remyelinate axons to promote electrical conduction (CaoQ, He Q, Wang Yet et al. Transplantation of ciliary neurotrophicfactor-expressing adult oligodendrocyte precursor cells promotesremyelination and functional recovery after spinal cord injury. J.Neurosci. 2010 30(8): 2989-3001).

AST-OPC1 is a population of oligodendrocyte progenitor cells (OPCs) thatare produced from human embryonic stem cells (hESCs) using a specificdifferentiation protocol (Nistor G I, Totoiu M O, Haque N, Carpenter MK, Keirstead H S. Human embryonic stem cells differentiate intooligodendrocytes in high purity and myelinate after spinal cordtransplantation. Glia. 2005 February; 49(3):385-96). AST-OPC1 has beencharacterized by the expression of several molecules that are associatedwith oligodendrocyte precursors, including Nestin and NG2. The cells arefurther characterized by their minimal or lack of expression of markersknown to be present in other cell types, such as neurons, astrocytes,endoderm, mesoderm, and hESCs (Keirstead H S, Nistor G, Bernal G, TotoiuM, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derivedoligodendrocyte progenitor cell transplants remyelinate and restorelocomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705; Zhang Y W, Denham J, Thies R S. Oligodendrocyteprogenitor cells derived from human embryonic stem cells expressneurotrophic factors. Stem Cells Dev. 2006 December; 15(6): 943-52). Invitro, AST-OPC1 also produces diffusible factors that support neuriteextension from sensory neurons (Zhang Y W, Denham J, Thies R S.Oligodendrocyte progenitor cells derived from human embryonic stem cellsexpress neurotrophic factors. Stem Cells Dev. 2006 December;15(6):943-52).

Pluripotent stem cell-derived neural cells have been used by researchersto treat CNS injuries and disorders in animal models. However, thereremain obstacles in the development of such therapies for clinicalapplications in humans. To date, there are no available therapiesutilizing human pluripotent stem cell-derived differentiated cellpopulations for the treatment of acute spinal cord injury or otherneurological conditions requiring CNS repair and/or remyelination.

SUMMARY

In various embodiments described herein, the present disclosureprovides, inter alia, a population of oligodendrocyte progenitor cells(OPCS) derived from pluripotent stem cells and methods of generating thesame for use in the treatment of acute spinal cord injury and otherconditions affecting the CNS. The present disclosure also identifies andprovides factors produced and secreted by the OPCS of the presentdisclosure that have capacity to augment neural repair. The presentdisclosure also provides methods and compositions for reducing spinalcord injury-induced parenchymal cavitation in a subject with a spinalcord injury.

In an embodiment, the present disclosure provides a container comprisinga composition comprising a population of allogeneic humanoligodendrocyte progenitor cells (OPCS) that are capable of engraftingat a spinal cord injury site of a human subject following implantationof a dose of the composition into the spinal cord injury site. Incertain embodiments, the OPCs do not elicit a humoral or cellular immuneresponse in the subject when the subject undergoes a low doseimmunosuppressant regimen.

In certain embodiments, the OPCs are capable of remaining within thespinal cord injury site of the subject for a period of about 180 days orlonger following implantation of a dose of the composition into thespinal cord injury site. In other embodiments, the OPCs are capable ofremaining within the spinal cord injury site of the subject for a periodof about 2 years or longer following implantation of a dose of thecomposition into the spinal cord injury site. In further embodiments,the OPCs are capable of remaining within the spinal cord injury site ofthe subject for a period of about 3 years or longer followingimplantation of a dose of the composition into the spinal cord injurysite. In yet further embodiments, the OPCs are capable of remainingwithin the spinal cord injury site of the subject for a period of about4 years or longer following implantation of a dose of the compositioninto the spinal cord injury site.

In certain embodiments, the OPCs are capable of forming a tissue matrixin the spinal cord injury site, thereby reducing spinal cordinjury-induced parenchymal cavitation. In certain embodiments, the OPCsare capable of forming a tissue matrix in the spinal cord injury sitewithin about 180 days or less.

In certain embodiments, the OPCs are capable of secreting one or morebiological factors. In certain embodiments, the biological factorssecreted by the OPCs of the present disclosure may promote, withoutlimitation, neural repair, axonal outgrowth and/or glialdifferentiation. In some embodiments, the OPCs are capable of secretingone or more of the factors selected from MCP-1, Clusterin, ApoE, TIMP1and TIMP2. In further embodiments the OPCs are capable of secretingMCP-1 and one or more factors selected from: Clusterin, ApoE, TIMP1 andTIMP2. In yet further embodiments, the OPCs are capable of secreting allof the factors MCP-1, Clusterin, ApoE, TIMP1 and TIMP2.

In certain embodiments, the present disclosure provides a containercomprising a composition comprising a population of allogeneicoligodendrocyte progenitor cells (OPCs) that are capable of engraftingat a spinal cord injury site of a human subject following implantationof a dose of the composition into the spinal cord injury site, whereinthe dose of the composition comprises between about 2×10⁶ and about50×10⁶ AST-OPC1. In some embodiments, the dose of the compositioncomprises about 50×10⁶ AST-OPC1. In some embodiments, the dose of thecomposition comprises about 40×10⁶ AS T-OPC 1. In some embodiments, thedose of the composition comprises about 30×10⁶ AS T-OPC 1. In someembodiments, the dose of the composition comprises about 20×10⁶AST-OPC1. In some embodiments, the dose of the composition comprisesabout 10×10⁶ AST-OPC1. In some embodiments, the dose of the compositioncomprises about 5×10⁶ AST-OPC1. In some embodiments, the dose of thecomposition comprises about 2×10⁶ AST-OPC1.

In certain embodiments, the present disclosure provides a containercomprising a composition comprising a population of allogeneicoligodendrocyte progenitor cells (OPCs) that are capable of presentingat a spinal cord injury site of a subject and are capable of producingno detectable systemic toxicity in the subject following implantation ofa dose of the composition into the spinal cord injury site. In certainembodiments, the OPCs do not induce significant alterations in ahematology, coagulation, urinalysis or clinical chemistry parameter ofthe subject.

In yet other embodiments, the present disclosure provides a method ofreducing spinal cord injury-induced parenchymal cavitation in a humansubject with an acute spinal cord injury, the method comprisingadministering to said subject a composition that comprises a populationof allogeneic oligodendrocyte progenitor cells (OPCs) that are capableof engrafting at a spinal cord injury site. In certain embodiments,administering the composition comprises directly injecting thecomposition into the spinal cord injury site approximately 5 mm caudalof the spinal cord injury epicenter. In certain embodiments, the methodfurther comprises administering to the subject a low doseimmunosuppressant regimen. In certain embodiments, the immunosuppressantregimen comprises a dose of tacrolimus at about 0.03 mg/kg/day per os,adjusted to maintain a trough blood concentration of about 3-7 ng/mLthrough about day 46 following the administering of the composition,followed by tapering off and discontinuing the immunosuppressant atabout day 60 following the administering of the composition comprising apopulation of allogeneically derived OPCs. In certain embodiments, theOPCs are capable of remaining within the spinal cord injury site of saidsubject for a period of about 180 days or longer following theadministration of the composition to the spinal cord injury site. Incertain embodiments, the OPCs are capable of remaining within the spinalcord injury site of said subject for a period of about 2 years or longerfollowing the administration of the composition to the spinal cordinjury site. In further embodiments the OPCs are capable of forming atissue matrix in the spinal cord injury site of said subject withinabout 180 days or less, thereby reducing spinal cord injury-inducedparenchymal cavitation. In certain embodiments, the subject has athoracic spinal cord injury. In other embodiments, the subject has acervical spinal cord injury. In certain embodiments, the compositioncomprises between about 2×10⁶ to about 50×10⁶ AST-OPC1 cells.

In additional embodiments, the present disclosure provides methods ofcharacterizing and verifying the purity of the OPCs of the presentdisclosure based on their marker expression profile. In otherembodiments, the present disclosure provides compositions and methodsfor stimulating axonal outgrowth in vitro using the OPCs of the presentdisclosure. In yet other embodiments, the present disclosure providescompositions and methods for stimulating axon myelination in vivo usingthe OPCs of the present disclosure. In further embodiments, the presentdisclosure provides methods for evaluating the safety and toxicity ofthe OPCs of the present disclosure in preclinical studies using rodentmodels of dysmyelination and contusion injury. In yet other embodiments,the present disclosure provides compositions and methods foradministering OPCs to human subjects with a spinal cord injury. In otherembodiments, the present disclosure provides compositions and methodsfor testing the safety and efficacy of administering OPCs to humansubjects with a spinal cord injury.

In additional embodiments, the present disclosure provides a populationof oligodendrocyte progenitor cells (OPCs) that are the in vitrodifferentiated progeny of pluripotent stem cells and that are suitablefor administering to a subject with a spinal cord injury. In certainembodiments, the population of OPCs are the in vitro differentiatedprogeny of human embryonic stem cells. In certain embodiments, theoligodendrocyte progenitor cells express one or more markers selectedfrom: Nestin, Olig 1, PDGF-Ra and NG2. In some embodiments, at least 70%of the cells in the OPC population are positive for Nestin expression.In other embodiments, at least 30% of the cells in the OPC populationare positive for NG2 expression.

In additional embodiments, the present disclosure provides a method fortreating a subject in need of therapy, comprising administering to thesubject a population of OPCs that are the in vitro differentiatedprogeny of pluripotent stem cells. In some embodiments, the method fortreating a subject in need of therapy comprises administering to thesubject a population of OPCs that are the in vitro differentiatedprogeny of human embryonic stem cells. In some embodiments, the subjectin need of therapy has a defect or injury requiring myelin repair orremyelination. In some embodiments, the subject in need of therapy has aspinal cord injury. In some embodiments, the subject in need of therapyis human.

In another embodiment, the present disclosure provides a pharmaceuticalcomposition comprising a population of oligodendrocyte progenitor cellsthat are the in vitro differentiated progeny of pluripotent stem cellsand a biologically acceptable carrier or delivery system.

BRIEF DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings.

FIG. 1A consists of bright-field photomicrographs of differentiation ofhuman embryonic stem cells into AST-OPC1 over 41 days.

FIG. 1B shows representative flow cytometry data for the neural/gliallineage markers Nestin and NG2 and the pluripotency marker Oct4 from theday 41 AST-OPC1.

FIGS. 2A-2B show representative images of rat primary cortical neuronscultured in control medium for 14 days (FIG. 2A) or co-cultured withAST-OPC1 from day 4 to day 14 (FIG. 2B) and labeled byimmunocytochemistry against the axonal marker SMI-312.

FIG. 2C depicts quantification of axon outgrowth in rat primary corticalneurons cultured with AST-OPC1 compared to vehicle control alone on day14.

FIGS. 2D-2E show high magnification photomicrographs of thoracic spinalcord from immunodeficient Rag2^(−/−)γc^(−/−)/shi^(−/−) mice transplantedwith AST-OPC1. By 2 months post-transplantation, myelinated fibers weredetectable within the graft site in close association with cellspositively labeled with the anti-human nuclei antibody (hNUC) (FIG. 2D),whereas no myelin staining was observed in the spinal cord outside theAST-OPC1 graft site (FIG. 2E).

FIGS. 3A-3D depict representative low and high magnificationphotomicrographs of the injury/graft site in a rat model of contusioninjury 9 months after thoracic spinal cord injury and injection ofvehicle (FIG. 3A and FIG. 3C) or transplantation of AST-OPC1 (FIG. 3Band FIG. 3D). At 9 months post-contusion, vehicle-treated animalsexhibited redirection of myelinated fibers around the lesion cavity(FIG. 3C), whereas cavitation was reduced in AST-OPC1-treated animals,and myelinated fibers were visible within the injury/graft site (FIG.3D).

FIG. 3E shows dot plot of cavitation area 9 months after thoracic spinalcord injury and vehicle or AST-OPC1 treatment. Asterisk denotessignificance relative to vehicle treatment based on two-tailed Student'st-test (p<0.05). Horizontal lines denote mean plus standard error of themean (SEM) for each treatment group (vehicle mean cavitationarea±SEM=1.29±0.372 mm², n=12; low-dose AST-OPC1 mean cavitationarea±SEM=0.378±0.201 mm², n=10, p=0.033; high-dose AST-OPC1 meancavitation area±SEM=0.244±0.111 mm² n=11, p=0.0125).

FIG. 3F shows representative photomicrograph of an adjacent tissuesection from the AST-OPC1-treated animal in FIGS. 3B and 3D, showingpositive labeling with a human Alu DNA repeat sequence probe by in situhybridization (brown nuclear signal, eosin counterstain) within the sameregion exhibiting myelinated fibers.

FIG. 4A depicts quantification of human cell survival in the centralnervous system in AST-OPC1 treated, spinal cord injured rats.

FIG. 4B shows histological assessment of the presence of human cells atand rostral to the injury site at 180 days post-administration asdetermined by immunohistochemistry using antibodies against humannuclear antigen (hNUC). Magnification=100×.

FIG. 5 depicts measurement of allodynia in spinal cord injured rats 9months after administration of AST-OPC1.

FIGS. 6A-6E depict cystic-like epithelial structures in within theAST-OPC1 graft site 6 months after administration, observed on 6 of the252 animals injected with AST-OPC1. In a photographic montage of thecontused spinal cord (FIG. 6A), brackets indicate the approximateboundaries of the highest graft density, and the cystic structure isindicated with a black arrow. To the left of the cystic structure, justcaudal to the indicated graft boundary, residual cavitation is apparent(FIG. 6B). The cystic structure was comprised of human cells based onpositive labeling with a human Alu DNA repeat sequence probe (brownnuclear signal, eosin counterstain (FIG. 6C). Few Ki-67-positive cellswere detectable by immunohistochemistry within the injury/graft site orwithin the cystic structure (FIG. 6D). Myelinated fibers labeled withEriochrome cyanine (blue) were detectable within the injury/graft siteand immediately adjacent to the cystic structure (FIG. 6E).

FIG. 7A and FIG. 7B depict frequency of teratoma formation in micetreated with AST-OPC1 or AST-OPC1 spiked with increasing levels ofundifferentiated hESCs.

FIGS. 8A-8D depict representative histology of rat thoracic spinal cord9 months after contusion injury and AST-OPC1 transplantation. Each ofthe FIGS. 8A-8D corresponds to a different animal.

FIG. 9 depicts phenotypic marker expression of AST-OPC1 as assessed byimmunocytochemistry.

FIG. 10 depicts factors secreted by AST-OPC1 with putative roles inneuronal repair.

FIG. 11 depicts physiological parameters examined to assess thepotential systemic toxicity of AST-OPC1.

FIG. 12A and FIG. 12B depict sagittal (FIG. 12A) and axial (FIG. 12B)MRI scans on T2 thoracic vertebra in a clinical trial subject at 3 yearsafter administration of 2×10⁶ AST-OPC1 to the subject. The images arerepresentative of 4 of the 5 enrolled clinical trial subjects.

FIG. 13 depicts the results of immune monitoring assays in the 5clinical trial subjects at successive time points up to one yearpost-grafting.

FIG. 14 depicts a summary of sensory neurological function in the 5clinical trial subjects from the baseline/beginning of clinical trialthrough year 2 (year 3 for one subject).

FIGS. 15A-15C depict AST-OP1 prevention of parenchymal cavitation inrats with cervical spinal cord contusion injury.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that the present disclosure is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims For example, featuresillustrated with respect to one embodiment may be incorporated intoother embodiments, and features illustrated with respect to a particularembodiment may be deleted from that embodiment. Thus, the disclosurecontemplates that in some embodiments of the disclosure, any feature orcombination of features set forth herein can be excluded or omitted. Inaddition, numerous variations and additions to the various embodimentssuggested herein will be apparent to those skilled in the art in lightof the instant disclosure, which do not depart from the instantdisclosure. In other instances, well-known structures, interfaces, andprocesses have not been shown in detail in order not to unnecessarilyobscure the invention. It is intended that no part of this specificationbe construed to effect a disavowal of any part of the full scope of theinvention. Hence, the following descriptions are intended to illustratesome particular aspects of the disclosure, and not to exhaustivelyspecify all permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The terminology used in thedescription of the disclosure herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of thedisclosure.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the disclosure described herein can be used inany combination. Moreover, the present disclosure also contemplates thatin some embodiments of the disclosure, any feature or combination offeatures set forth herein can be excluded or omitted.

Methods disclosed herein can comprise one or more steps or actions forachieving the described method. The method steps and/or actions may beinterchanged with one another without departing from the scope of thepresent invention. In other words, unless a specific order of steps oractions is required for proper operation of the embodiment, the orderand/or use of specific steps and/or actions may be modified withoutdeparting from the scope of the present invention.

As used in the description of the disclosure and the appended claims,the singular forms “a,” “an” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The terms “about” and “approximately” as used herein when referring to ameasurable value such as a percentages, density, volume and the like, ismeant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about Xand Y” should be interpreted to include X and Y. As used herein, phrasessuch as “between about X and Y” mean “between about X and about Y” andphrases such as “from about X to Y” mean “from about X to about Y.”

The term “AST-OPC1” refers to a specific, characterized, in vitrodifferentiated cell population containing a mixture of oligodendrocyteprogenitor cells (OPCs) and other characterized cell types obtained fromundifferentiated human embryonic stem cells (uhESCs) according tospecific differentiation protocols disclosed herein.

Compositional analysis of AST-OPC1 by immunocytochemistry (ICC), flowcytometry, and quantitative polymerase chain reaction (qPCR)demonstrates that the cell population is comprised primarily of neurallineage cells of the oligodendrocyte phenotype. Other neural lineagecells, namely astrocytes and neurons, are present at low frequencies.The only non-neural cells detected in the population are epithelialcells. Mesodermal, endodermal lineage cells and uhESCs are routinelybelow quantitation or detection of the assays.

The term “oligodendrocyte progenitor cells” (OPCs), as used herein,refers to cells of neuroectoderm/glial lineage having thecharacteristics of a cell type found in the central nervous system,capable of differentiating into oligodendrocytes. These cells typicallyexpress the characteristic markers Nestin, NG2 and PDGF-Ra.

The terms “treatment,” “treat” “treated,” or “treating,” as used herein,can refer to both therapeutic treatment or prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological condition, symptom, disorder or disease, or toobtain beneficial or desired clinical results. In some embodiments, theterm may refer to both treating and preventing. For the purposes of thisdisclosure, beneficial or desired clinical results may include, but arenot limited to one or more of the following: alleviation of symptoms;diminishment of the extent of the condition, disorder or disease;stabilization (i.e., not worsening) of the state of the condition,disorder or disease; delay in onset or slowing of the progression of thecondition, disorder or disease; amelioration of the condition, disorderor disease state; and remission (whether partial or total), whetherdetectable or undetectable, or enhancement or improvement of thecondition, disorder or disease. Treatment includes eliciting aclinically significant response. Treatment also includes prolongingsurvival as compared to expected survival if not receiving treatment.

The term “subject,” as used herein includes, but is not limited to,humans, non-human primates and non-human vertebrates such as wild,domestic and farm animals including any mammal, such as cats, dogs,cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. Insome embodiments, the term “subject,” refers to a male. In someembodiments, the term “subject,” refers to a female.

As used herein, “implantation” or “transplantation” refers to theadministration of a cell population into a target tissue using asuitable delivery technique, (e.g., using an injection device).

As used herein, “engraftment” and “engrafting” refer to incorporation ofimplanted tissue or cells (i.e. “graft tissue” or “graft cells”) intothe body of a subject. The presence of graft tissue or graft cells at ornear the implantation site 180 days or later, post implantation, isindicative of engraftment. In certain embodiments, imaging techniques(such as, e.g. MRI imaging), can be used to detect the presence of grafttissue.

As used herein, “allogeneic” and “allogeneically derived” refer to cellpopulations derived from a source other than the subject and hencegenetically non-identical to the subject. In certain embodiments,allogeneic cell populations are derived from cultured pluripotent stemcells. In certain embodiments, allogeneic cell populations are derivedfrom hESCs. In other embodiments, allogeneic cell populations arederived from induced pluripotent stem (iPS) cells. In yet otherembodiments, allogeneic cell populations are derived from primatepluripotent (pPS) cells.

As used herein, “parenchymal cavitation” refers to formation of a lesionor cavity within a CNS injury site or proximate to a CNS injury site, inan area normally occupied by parenchymal CNS tissue. The cavities orlesions can be filled with extracellular fluid and may containmacrophages, small bands of connective tissue and blood vessels.

The terms “central nervous system” and “CNS” as used interchangeablyherein refer to the complex of nerve tissues that control one or moreactivities of the body, which include but are not limited to, the brainand the spinal cord in vertebrates.

Propagation and Culture of Undifferentiated Pluripotent Stem Cells

In certain embodiments, the present disclosure provides methods toproduce large numbers of highly pure, characterized oligodendrocyteprogenitor cells from pluripotent stem cells. Derivation ofoligodendrocyte progenitor cells (OPCs) from pluripotent stem cellsaccording to the methods of the invention provides a renewable andscalable source of OPCs for a number of important therapeutic, research,development, and commercial purposes, including treatment of acutespinal cord injury.

Methods of propagation and culture of undifferentiated pluripotent stemcells have been previously described. With respect to tissue and cellculture of pluripotent stem cells, the reader may wish to refer to anyof numerous publications available in the art, e.g., Teratocarcinomasand Embryonic Stem cells: A Practical Approach (E. J. Robertson, Ed.,IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M.Wasserman et al., Eds., Academic Press 1993); Embryonic Stem CellDifferentiation in vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993);Properties and Uses of Embryonic Stem Cells: Prospects for Applicationto Human Biology and Gene Therapy (P. D. Rathj en et al., Reprod.Fertil. Dev. 10:31, 1998; and R. I. Freshney, Culture of Animal Cells,Wiley-Liss, New York, 2000).

In certain embodiments, a method can be carried out on a pluripotentstem cell line. In other embodiments, a method can be carried out on anembryonic stem cell line. In an embodiment, a method can be carried outon a plurality of undifferentiated stem cells that are derived from anH1, H7, H9, H13, or H14 cell line. In another embodiment,undifferentiated stem cells can be derived from an induced pluripotentstem cell (iPS) line. In another embodiment, a method can be carried outon a primate pluripotent stem (pPS) cell line. In yet anotherembodiment, undifferentiated stem cells can be derived from parthenotes,which are embryos stimulated to produce hESCs without fertilization.

In one embodiment, undifferentiated pluripotent stem cells can bemaintained in an undifferentiated state without added feeder cells (see,e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free culturesare typically supported by a nutrient medium containing factors thatpromote proliferation of the cells without differentiation (see, e.g.,U.S. Pat. No. 6,800,480). In one embodiment, conditioned mediacontaining such factors can be used. Conditioned media can be obtainedby culturing the media with cells secreting such factors. Suitable cellsinclude, but are not limited to, irradiated (˜4,000 Rad) primary mouseembryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-likecells derived from pPS cells (U.S. Pat. No. 6,642,048). Medium can beconditioned by plating the feeders in a serum free medium, such asknock-out DMEM supplemented with 20% serum replacement and 4 ng/mL bFGF.Medium that has been conditioned for 1-2 days can be supplemented withfurther bFGF, and used to support pPS cell culture for 1-2 days (see.e.g., WO 01/51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (such as, e.g., a fibroblast growthfactor or forskolin) that promote proliferation of the cells in anundifferentiated form. Non-limiting examples include a base medium likeX-VIVO™ 10 (Lonza, Walkersville, Md.) or QBSF™-60 (Quality BiologicalInc. Gaithersburg, Md.), supplemented with bFGF at 40-80 ng/mL, andoptionally containing SCF (15 ng/mL), or Flt3 ligand (75 ng/mL) (see,e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulationshave the advantage of supporting cell growth at 2-3 times the rate inother systems (see, e.g., WO 03/020920). In one embodiment,undifferentiated pluripotent cells such as hESCs, can be cultured in amedia comprising bFGF and TGFβ. Non-limiting example concentrations ofbFGF include about 80 ng/ml. Non-limiting example concentrations of TGFβinclude about 0.5 ng/ml.

In one embodiment, undifferentiated pluripotent cells can be cultured ona layer of feeder cells, typically fibroblasts derived from embryonic orfetal tissue (Thomson et al. (1998) Science 282:1145). Feeder cells canbe derived, inter alia, from a human or a murine source. Human feedercells can be isolated from various human tissues, or can be derived viadifferentiation of human embryonic stem cells into fibroblast cells(see, e.g., WO 01/51616). In one embodiment, human feeder cells that canbe used include, but are not limited to, placental fibroblasts (see,e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tubeepithelial cells (see, e.g., Richards et al. (2002) Nat. Biotechnol.,20:933), foreskin fibroblasts (see, e.g., Amit et al. (2003) Biol.Reprod.68:2150), and uterine endometrial cells (see, e.g., Lee et al.(2005) Biol. Reprod. 72(1):42).

Various solid surfaces can be used in the culturing of undifferentiatedpluripotent cells. Those solid surfaces include, but are not limited to,standard commercially available cell culture plates, such as 6-well,24-well, 96-well, or 144-well plates. Other solid surfaces include, butare not limited to, microcarriers and disks. Solid surfaces suitable forgrowing undifferentiated pluripotent cells can be made of a variety ofsubstances including, but not limited to, glass or plastic such aspolystyrene, polyvinylchloride, polycarbonate, polytetrafluorethylene,melinex, thermanox, or combinations thereof. In one embodiment, suitablesurfaces can comprise one or more polymers, such as, e.g., one or moreacrylates. In one embodiment, a solid surface can be three-dimensionalin shape. Non-limiting examples of three-dimensional solid surfaces aredescribed, e.g., in U.S. Patent Pub. No. 2005/0031598.

In one embodiment, undifferentiated stem cells can be grown underfeeder-free conditions on a growth substrate. In one embodiment, agrowth substrate can be Matrigel® (e.g., Matrigel® or Matrigel® GFR),recombinant Laminin, or Vitronectin. In another embodiment,undifferentiated stem cells can be subcultured using various methodssuch as using collagenase, or such as manual scraping. In anotherembodiment, undifferentiated stem cells can be subcultured usingnon-enzymatic means, such as 0.5 mM EDTA in PBS, or such as usingReLeSR™. In an embodiment, a plurality of undifferentiated stem cellsare seeded or subcultured at a seeding density that allows the cells toreach confluence in about three to about ten days. In an embodiment, theseeding density can range from about 6.0×10³ cells/cm² to about 5.0×105cells/cm², such as about 1.0×10⁴ cells/cm², such as about 5.0×10⁴cells/cm², such as about 1.0×10⁵ cells/cm², or such as about 3.0×10⁵cells/cm² of growth surface. In another embodiment, the seeding densitycan range from about 6.0×10³ cells/cm² to about 1.0×104 cells/cm² ofgrowth surface, such as about 6.0×10³ cells/cm² to about 9.0×10³cells/cm², such as about 7.0×10³ cells/cm² to about 1.0×10⁴ cells/cm²,such as about 7.0×10³ cells/cm² to about 9.0×10³ cells/cm², or such asabout 7.0×10³ cells/cm² to about 8.0×10³ cells/cm² of growth surface. Inyet another embodiment the seeding density can range from about 1.0×10⁴cells/cm² to about 1.0×10⁵ cells/cm² of growth surface, such as about2.0×10⁴ cells/cm² to about 9.0×10⁴ cells/cm², such as about 3.0×10⁴cells/cm² to about 8.0×10⁴ cells/cm², such as about 4.0×10⁴ cells/cm² toabout 7.0×10⁴ cells/cm², or such as about 5.0×10⁴ cells/cm² to about6.0×10⁴ cells/cm² of growth surface. In an embodiment, the seedingdensity can range from about 1.0×10⁵ cells/cm² to about 5.0×10⁵cells/cm² of growth surface, such as about 1.0×10⁵ cells/cm² to about4.5×10⁵ cells/cm², such as about 1.5×10⁵ cells/cm² to about 4.0×10⁵cells/cm², such as about 2.0×10⁵ cells/cm² to about 3.5×10⁵ cells/cm²,or such as about 2.5×10⁵ cells/cm² to about 3.0×10⁵ cells/cm² of growthsurface.

Any of a variety of suitable cell culture and sub-culturing techniquescan be used to culture cells in accordance with the present disclosure.For example, in one embodiment, a culture medium can be exchanged at asuitable time interval. In one embodiment, a culture medium can becompletely exchanged daily, initiating about 2 days after sub-culturingof the cells. In another embodiment, when a culture reaches about 90%colony coverage, a surrogate flask can be sacrificed and enumeratedusing one or more suitable reagents, such as, e.g., Collagenase IV and0.05% Trypsin-EDTA in series to achieve a single cellsuspension forquantification. In an embodiment, a plurality undifferentiated stemcells can then be sub-cultured before seeding the cells on a suitablegrowth substrate (e.g., Matrigel® GFR) at a seeding density that allowsthe cells to reach confluence over a suitable period of time, such as,e.g., in about three to ten days. In one embodiment, undifferentiatedstem cells can be subcultured using Collagenase IV and expanded on arecombinant laminin matrix. In one embodiment, undifferentiated stemcells can be subcultured using Collagenase IV and expanded on aMatrigel® matrix. In one embodiment, undifferentiated stem cells can besubcultured using ReLeSR™ and expanded on a Vitronectin matrix.

In one embodiment, the seeding density can range from about 6.0×103cells/cm² to about 5.0×10⁵ cells/cm², such as about 1.0×104 cells/cm²,such as about 5.0×104 cells/cm², such as about 1.0×10⁵ cells/cm², orsuch as about 3.0×105 cells/cm² of growth surface. In anotherembodiment, the seeding density can range from about 6.0×103 cells/cm²to about 1.0×10⁴ cells/cm² of growth surface, such as about 6.0×10³cells/cm² to about 9.0×10³ cells/cm², such as about 7.0×10³ cells/cm² toabout 1.0×10⁴ cells/cm², such as about 7.0×10³ cells/cm² to about9.0×10³ cells/cm², or such as about 7.0×10³ cells/cm² to about 8.0×10³cells/cm² of growth surface. In yet another embodiment, the seedingdensity can range from about 1.0×10⁴ cells/cm² to about 1.0×10⁵cells/cm² of growth surface, such as about 2.0×10⁴ cells/cm² to about9.0×10⁴ cells/cm², such as about 3.0×10⁴ cells/cm² to about 8.0×10⁴cells/cm², such as about 4.0×10⁴ cells/cm² to about 7.0×10⁴ cells/cm²,or such as about 5.0×10⁴ cells/cm² to about 6.0×10⁴ cells/cm² of growthsurface. In an embodiment, the seeding density can range from about1.0×10⁵ cells/cm² to about 5.0×10⁵ cells/cm² of growth surface, such asabout 1.0×10⁵ cells/cm² to about 4.5×10⁵ cells/cm², such as about1.5×10⁵ cells/cm² to about 4.0×10⁵ cells/cm², such as about 2.0×10⁵cells/cm² to about 3.5×10⁵ cells/cm², or such as about 2.5×10⁵ cells/cm²to about 3.0×10⁵ cells/cm² of growth surface.

Oligodendrocyte Progenitor Cell Compositions

As discussed above, the present disclosure provides compositionscomprising a population of oligodendrocyte progenitor cells (OPCs) aswell as methods of making and using the same from use in the treatmentof acute spinal cord injury and other related CNS conditions. In certainembodiments, the OPCs of the present disclosure are capable of producingand secreting one or more biological factors that may augment neuralrepair.

In one embodiment, a cell population can have a common geneticbackground. In an embodiment, a cell population may be derived from onehost. In an embodiment, a cell population can be derived from apluripotent stem cell line. In another embodiment, a cell population canbe derived from an embryonic stem cell line. In an embodiment, a cellpopulation can be derived from a hESC line. In an embodiment, a hESCline can be an H1, H7, H9, H13, or H14 cell line. In another embodiment,a cell population can be derived from an induced pluripotent stem cell(iPS) line. In an embodiment a cell population can be derived from asubject in need thereof (e.g., a cell population can be derived from asubject that is in need to treatment). In yet another embodiment, a hESCline can be derived from parthenotes, which are embryos stimulated toproduce hESCs without fertilization.

In certain embodiments, the OPCs of the present disclosure express oneor more markers chosen from Nestin, NG2, Olig 1 and PDGF-Ra. In certainembodiments, the OPCs of the present disclosure express all of themarkers Nestin, NG2, Olig 1 and PDGF-Ra. In some embodiments, at least70% of AST-OPC1 are positive for Nestin expression. In some embodiments,at least 30% of AST-OPC1 are positive for NG2 expression. In someembodiments, at least 70% of AST-OPC1 are positive for Olig 1expression. In some embodiments, at least 70% of AST-OPC1 are positivefor PDGF-Ra expression. The specific markers and combinations of variousmarkers expressed by the cell populations of the present disclosure canbe determined and quantified, for example, by flow cytometry.Non-limiting examples of the markers expressed by the cells of thepresent disclosure are provided in FIG. 9.

In certain embodiments, the OPCs of the present disclosure are capableof secreting one or more biological factors. In certain embodiments, theone or more biological factors secreted by the OPCs of the presentdisclosure may promote, without limitation, neural repair, axonaloutgrowth and/or glial differentiation, or any combination thereof. Insome embodiments, the OPCs are capable of secreting one or more factorsthat stimulate axonal outgrowth. In some embodiments, the OPCs arecapable of secreting one or more factors promoting glial differentiationby neural precursor cells. In some embodiments, the OPCs are capable ofsecreting one or more chemoattractants for neural precursor cells. Insome embodiments, the OPCs are capable of secreting one or moreinhibitors of matrix metalloproteinases. In some embodiments, the OPCsare capable of secreting one or more factors inhibiting cell death afterspinal cord injury. In some embodiments, the OPCs are capable ofsecreting one or more factors that are upregulated post-cellular injuryand that aid in the clearance of misfolded proteins.

In certain embodiments, the OPCs are capable of producing and secretingone or more biological factors selected from MCP-1, Clusterin, ApoE,TIMP1 and TIMP2. In further embodiments the OPCs are capable ofproducing and secreting MCP-1 and one or more of the factors selectedfrom Clusterin, ApoE, TIMP1 and TIMP2. In yet further embodiments, theOPCs are capable of producing and secreting all of the factors MCP-1,Clusterin, ApoE, TIMP1 and TIMP2.

In an embodiment, a biological factor can be secreted by a compositioncomprising a population of OPCs at a concentration of more than about 50pg/ml, such as more than about 100 pg/ml, such as more than about 200pg/ml, such as more than about 300 pg/ml, such as more than about 400pg/ml, such as more than about 500 pg/ml, such as more than about 1,000pg/ml, such as more than about 2,000 pg/ml, such as more than about3,000 pg/ml, such as more than about 4,000 pg/ml, such as more thanabout 5,000 pg/ml, such as more than about 6,000 pg/ml, or such as morethan about 7,000 pg/ml. In certain embodiments, a biological factor canbe secreted by a composition comprising a population of cells comprisingOPCs at a concentration ranging from about 50 pg/ml to about 100,000pg/ml, such as about 100 pg/ml, such as about 150 pg/ml, such as about200 pg/ml, such as about 250 pg/ml, such as about 300 pg/ml, such asabout 350 pg/ml, such as about 400 pg/ml, such as about 450 pg/ml, suchas about 500 pg/ml, such as about 550 pg/ml, such as about 600 pg/ml,such as about 650 pg/ml, such as about 700 pg/ml, such as about 750pg/ml, such as about 800 pg/ml, such as about 850 pg/ml, such as about900 pg/ml, such as about 1,000 pg/ml, such as about 1,500 pg/ml, such asabout 2,000 pg/ml, such as about 2,500 pg/ml, such as about 3,000 pg/ml,such as about 3,500 pg/ml, such as about 4,000 pg/ml, such as about4,500 pg/ml, such as about 5,000 pg/ml, such as about 5,500 pg/ml, suchas about 6,000 pg/ml, such as about 6,500 pg/ml, such as about 7,000pg/ml, such as about 7,500 pg/ml, such as about 8,000 pg/ml, such asabout 8,500 pg/ml, such as about 9,000 pg/ml, such as about 10,000pg/ml, such as about 15,000 pg/ml, such as about 20,000 pg/ml, such asabout 25,000 pg/ml, such as about 30,000 pg/ml, such as about 35,000pg/ml, such as about 40,000 pg/ml, such as about 45,000 pg/ml, such asabout 50,000 pg/ml, such as about 55,000 pg/ml, such as about 60,000pg/ml, such as about 65,000 pg/ml, such as about 70,000 pg/ml, such asabout 75,000 pg/ml, such as about 80,000 pg/ml, such as about 85,000pg/ml, such as about 90,000 pg/ml, such as about 95,000 pg/ml.

In certain embodiments, a biological factor can be secreted by acomposition comprising a population of cells comprising OPCs at aconcentration ranging from about 1,000 pg/ml to about 10,000 pg/ml, suchas about 1,000 pg/ml to about 2,000 pg/ml, such as about 2,000 pg/ml toabout 3,000 pg/ml, such as about 3,000 pg/ml to about 4,000 pg/ml, suchas about 4,000 pg/ml to about 5,000 pg/ml, such as about 5,000 pg/ml toabout 6,000 pg/ml, such as about 6,000 pg/ml to about 7,000 pg/ml, suchas about 7,000 pg/ml to about 8,000 pg/ml, such as about 8,000 pg/ml toabout 9,000 pg/ml, or such as about 9,000 pg/ml to about 10,000 pg/ml.

In certain embodiments, a biological factor can be secreted by acomposition comprising a population of cells comprising OPCs at aconcentration ranging from about 10,000 pg/ml to about 100,000 pg/ml,such as about 10,000 pg/ml to about 20,000 pg/ml, such as about 20,000pg/ml to about 30,000 pg/ml, such as about 30,000 pg/ml to about 40,000pg/ml, such as about 40,000 pg/ml to about 50,000 pg/ml, such as about50,000 pg/ml to about 60,000 pg/ml, such as about 60,000 pg/ml to about70,000 pg/ml, such as about 70,000 pg/ml to about 80,000 pg/ml, such asabout 80,000 pg/ml to about 90,000 pg/ml, or such as about 90,000 pg/mlto about 100,000 pg/ml.

In some embodiments, Clusterin can be secreted by a compositioncomprising a population of cells comprising OPCs at a concentrationranging from about 1,000 pg/ml to about 100,000 pg/ml. In certainembodiments, Clusterin can be secreted by a composition comprising apopulation of cells comprising OPCs at a concentration ranging fromabout 10,000 pg/ml to about 50,000 pg/ml. In some embodiments, MCP-1 canbe secreted by a composition comprising a population of cells comprisingOPCs at a concentration ranging from about 500 pg/ml to about 50,000pg/ml. In certain embodiments, MCP-1 can be secreted by a compositioncomprising a population of cells comprising OPCs at a concentrationranging from about 5,000 pg/ml to about 15,000 pg/ml. In someembodiments, ApoE can be secreted by a composition comprising apopulation of cells comprising OPCs at a concentration ranging fromabout 100 pg/ml to about 10,000 pg/ml. In certain embodiments, ApoE canbe secreted by a composition comprising a population of cells comprisingOPCs at a concentration ranging from about 500 pg/ml to about 5,000pg/ml. In some embodiments, TIMP1 can be secreted by a compositioncomprising a population of cells comprising OPCs at a concentrationranging from about 100 pg/ml to about 10,000 pg/ml. In certainembodiments, TIMP1 can be secreted by a composition comprising apopulation of cells comprising OPCs at a concentration ranging fromabout 500 pg/ml to about 5,000 pg/ml. In some embodiments, TIMP2 can besecreted by a composition comprising a population of cells comprisingOPCs at a concentration ranging from about 100 pg/ml to about 10,000pg/ml. In certain embodiments, TIMP2 can be secreted by a compositioncomprising a population of cells comprising OPCs at a concentrationranging from about 500 pg/ml to about 5,000 pg/ml.

Pharmaceutical Compositions

The OPCs of the present disclosure can be administered to a subject inneed of therapy per se. Alternatively, the cells of the presentdisclosure can be administered to the subject in need of therapy in apharmaceutical composition mixed with a suitable carrier and/or using adelivery system.

As used herein, the term “pharmaceutical composition” refers to apreparation comprising a therapeutic agent or therapeutic agents incombination with other components, such as physiologically suitablecarriers and excipients.

As used herein, the term “therapeutic agent” can refer to the cells ofthe present disclosure accountable for a biological effect in thesubject. Depending on the embodiment of the disclosure, “therapeuticagent” can refer to the oligodendrocyte progenitor cells of thedisclosure. Alternatively, “therapeutic agent” can refer to one or morefactors secreted by the oligodendrocyte progenitor cells of thedisclosure. Non-limiting examples of secreted factors are listed in FIG.10.

As used herein, the terms “carrier”, “pharmaceutically acceptablecarrier” and “biologically acceptable carrier” may be usedinterchangeably and refer to a diluent or a carrier substance that doesnot cause significant adverse effects or irritation in the subject anddoes not abrogate the biological activity or effect of the therapeuticagent. In certain embodiments, a pharmaceutically acceptable carrier cancomprise dimethyl sulfoxide (DMSO). In other embodiments, apharmaceutically acceptable carrier does not comprise dimethylsulfoxide. The term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of thetherapeutic agent.

The therapeutic agent or agents of the present disclosure can beadministered as a component of a hydrogel, such as those described inU.S. patent application Ser. No. 14/275,795, filed May 12, 2014, andU.S. Pat. Nos. 8,324,184 and 7,928,069.

The compositions in accordance with the present disclosure can beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can containformulatory agents such as suspending, stabilizing and/or dispersingagents. In certain embodiments, the compositions can be formulated to beadapted for cryopreservation.

The compositions in accordance with the present disclosure can beformulated for administration via a direct injection to the spinal cordof a subject. In certain embodiments, a composition in accordance withthe present disclosure can be formulated for intracerebral,intraventricular, intrathecal, intranasal, or intracisternaladministration to a subject. In certain embodiments, a composition inaccordance with the present disclosure can be formulated foradministration via an injection directly into or immediately adjacent toan infarct cavity in the brain of a subject. In certain embodiments, acomposition in accordance with the present disclosure can be formulatedfor administration through implantation. In certain embodiments, acomposition in accordance with the present disclosure can be formulatedas a solution.

In certain embodiments, a composition in accordance with the presentdisclosure can comprise from about 1×10⁶ to about 5×10⁸ cells permilliliter, such as about 1×106 cells per milliliter, such as about2×10⁶ cells per milliliter, such as about 3×10⁶ cells per milliliter,such as about 4×10⁶ cells per milliliter, such as about 5×10⁶ cells permilliliter, such as about 6×10⁶ cells per milliliter, such as about7×10⁶ cells per milliliter, such as about 8×10⁶ cells per milliliter,such as about 9×10⁶ cells per milliliter, such as about 1×10⁷ cells permilliliter, such as about 2×10⁷ cells per milliliter, such as about3×10⁷ cells per milliliter, such as about 4×10⁷ cells per milliliter,such as about 5×10⁷ cells per milliliter, such as about 6×10⁷ cells permilliliter, such as about 7×10⁷ cells per milliliter, such as about8×10⁷ cells per milliliter, such as about 9×107 cells per milliliter,such as about 1×10⁸ cells per milliliter, such as about 2×10⁸ cells permilliliter, such as about 3×10⁸ cells per milliliter, such as about4×10⁸ cells per milliliter, or such as about 5×10⁸ cells per milliliter.In certain embodiments, a composition in accordance with the presentdisclosure can comprise from about 1×10⁸ to about 5×10⁸ cells permilliliter, such as about 1×10⁸ to about 4×10⁸ cells per milliliter,such as about 2×10⁸ to about 5×10⁸ cells per milliliter, such as about1×10⁸ to about 3×10⁸ cells per milliliter, such as about 2×10⁸ to about4×10⁸ cells per milliliter, or such as about 3×10⁸ to about 5×10⁸ cellsper milliliter. In yet another embodiment, a composition in accordancewith the present disclosure can comprise from about 1×10⁷ to about 1×10⁸cells per milliliter, such as about 2×10⁷ to about 9×10⁷ cells permilliliter, such as about 3×10⁷ to about 8×10⁷ cells per milliliter,such as about 4×10⁷ to about 7×10⁷ cells per milliliter, or such asabout 5×10⁷ to about 6×10⁷ cells per milliliter. In an embodiment, acomposition in accordance with the present disclosure can comprise fromabout 1×10⁶ to about 1×10⁷ cells per milliliter, such as about 2×10⁶ toabout 9×10⁶ cells per milliliter, such as about 3×10⁶ to about 8×10⁶cells per milliliter, such as about 4×10⁶ to about 7×10⁶ cells permilliliter, or such as about 5×10⁶ to about 6×10⁶ cells per milliliter.In yet another embodiment, a composition in accordance with the presentdisclosure can comprise at least about 1×10⁶ cells per milliliter, suchas at least about 2×10⁶ cells per milliliter, such as at least about3×10⁶ cells per milliliter, such as at least about 4×10⁶ cells permilliliter, such as at least about 5×10⁶ cells per milliliter, such asat least about 6×10⁶ cells per milliliter, such as at least about 7×10⁶cells per milliliter, such as at least about 8×10⁶ cells per milliliter,such as at least about 9×10⁶ cells per milliliter, such as at leastabout 1×10⁷ cells per milliliter, such as at least about 2×10⁷ cells permilliliter, such as at least about 3×10⁷ cells per milliliter, such asat least about 4×10⁷ cells per milliliter, or such as at least about5×10⁷ cells per milliliter. In an embodiment, a composition inaccordance with the present disclosure can comprise up to about 1×10⁸cells or more, such as up to about 2×10⁸ cells per milliliter or more,such as up to about 3×10⁸ cells per milliliter or more, such as up toabout 4×10⁸ cells per milliliter or more, such as up to about 5×10⁸cells per milliliter or more, or such as up to about 6×10⁸ cells permilliliter.

In an embodiment, a composition in accordance with the presentdisclosure can comprise from about 4×10⁷ to about 2×10⁸ cells permilliliter.

In yet another embodiment, a composition in accordance with the presentdisclosure can have a volume ranging from about 10 microliters to about5 milliliters, such as about 20 microliters, such as about 30microliters, such as about 40 microliters, such as about 50 microliters,such as about 60 microliters, such as about 70 microliters, such asabout 80 microliters, such as about 90 microliters, such as about 100microliters, such as about 200 microliters, such as about 300microliters, such as about 400 microliters, such as about 500microliters, such as about 600 microliters, such as about 700microliters, such as about 800 microliters, such as about 900microliters, such as about 1 milliliter, such as about 1.5 milliliters,such as about 2 milliliters, such as about 2.5 milliliters, such asabout 3 milliliters, such as about 3.5 milliliters, such as about 4milliliters, or such as about 4.5 milliliters. In an embodiment, acomposition in accordance with the present disclosure can have a volumeranging from about 10 microliters to about 100 microliters, such asabout 20 microliters to about 90 microliters, such as about 30microliters to about 80 microliters, such as about 40 microliters toabout 70 microliters, or such as about 50 microliters to about 60microliters. In another embodiment, a composition in accordance with thepresent disclosure can have a volume ranging from about 100 microlitersto about 1 milliliter, such as about 200 microliters to about 900microliters, such as about 300 microliters to about 800 microliters,such as about 400 microliters to about 700 microliters, or such as about500 microliters to about 600 microliters. In yet another embodiment, acomposition in accordance with the present disclosure can have a volumeranging from about 1 milliliter to about 5 milliliters, such as about 2milliliter to about 5 milliliters, such as about 1 milliliter to about 4milliliters, such as about 1 milliliter to about 3 milliliters, such asabout 2 milliliter to about 4 milliliters, or such as about 3 milliliterto about 5 milliliters. In an embodiment, a composition in accordancewith the present disclosure can have a volume of about 20 microliters toabout 500 microliters. In another embodiment, a composition inaccordance with the present disclosure can have a volume of about 50microliters to about 100 microliters. In yet another embodiment, acomposition in accordance with the present disclosure can have a volumeof about 50 microliters to about 200 microliters. In another embodiment,a composition in accordance with the present disclosure can have avolume of about 20 microliters to about 400 microliters.

In certain embodiments, the present disclosure provides a containercomprising a composition comprising a population of OPCs derived inaccordance with one or more methods of the present disclosure. Incertain embodiments, a container can be configured for cryopreservation.In certain embodiments, a container can be configured for administrationto a subject in need thereof. In certain embodiments, a container can bea prefilled syringe.

For general principles in medicinal formulation, the reader is referredto Allogeneic Stem Cell Transplantation, Lazarus and Laughlin Eds.Springer Science+ Business Media LLC 2010; and Hematopoietic Stem CellTherapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.Choice of the cellular excipient and any accompanying elements of thecomposition will be adapted in accordance with the route and device usedfor administration. In certain embodiments, the composition can alsocomprise or be accompanied by one or more other ingredients thatfacilitate the engraftment or functional mobilization of the enrichedtarget cells. Suitable ingredients can include matrix proteins thatsupport or promote adhesion of the target cell type or that promotevascularization of the implanted tissue.

Uses of the Cells of the Present Disclosure

In various embodiments as described herein, the present disclosureprovides methods of using a cell population that comprises pluripotentstem cell-derived OPCs for improving one or more neurological functionsin a subject in need of therapy. In certain embodiments, methods forusing pluripotent stem-cell derived OPCs in the treatment of acutespinal cord injury are provided. In other embodiments, methods for usingpluripotent stem-cell derived OPCs in the treatment of other traumaticCNS injuries are provided. In other embodiments, methods for usingpluripotent stem-cell derived OPCs in the treatment of non-traumatic CNSdisorders or conditions are provided. In certain embodiments, a cellpopulation in accordance with the present disclosure can be injected orimplanted into a subject in need thereof.

In certain embodiments, methods for using pluripotent stem-cell derivedOPCs in the treatment of conditions requiring myelin repair orremyelination are provided. The following are non-limiting examples ofconditions, diseases and pathologies requiring myelin repair orremyelination: multiple sclerosis, the leukodystrophies, theGuillain-Barre Syndrome, the Charcot-Marie-Tooth neuropathy, Tay-Sachsdisease, Niemann-Pick disease, Gaucher disease and Hurler syndrome.Other conditions that result in demyelination include but are notlimited to inflammation, stroke, immune disorders, metabolic disordersand nutritional deficiencies (such as lack of vitamin B12). The OPCs ofthe present disclosure can also be used for myelin repair orremyelination in traumatic injuries resulting in loss of myelination,such as acute spinal cord injury.

The OPCs are administered in a manner that permits them to graft ormigrate to the intended tissue site and reconstitute or regenerate thefunctionally deficient area. Administration of the cells can be achievedby any method known in the art. For example the cells can beadministered surgically directly to the organ or tissue in need of acellular transplant. Alternatively non-invasive procedures can be usedto administer the cells to the subject. Non-limiting examples ofnon-invasive delivery methods include the use of syringes and/orcatheters to deliver the cells into the organ or tissue in need ofcellular therapy.

The subject receiving the OPCs of the present disclosure may be treatedto reduce immune rejection of the transplanted cells. Methodscontemplated include the administration of traditional immunosuppressivedrugs such as, e.g., tacrolimus, cyclosporin A (Dunn et al., Drugs61:1957, 2001), or inducing immunotolerance using a matched populationof pluripotent stem cell-derived cells (WO 02/44343; U.S. Pat. No.6,280,718; WO 03/050251). Alternatively a combination ofanti-inflammatory (such as prednisone) and immunosuppressive drugs canbe used. The OPCs of the invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration.

Use in treatment of CNS traumatic injury. In certain embodiments, a cellpopulation in accordance with the present disclosure can be capable ofengrafting at a spinal cord injury site following implantation of acomposition comprising the cell population into the spinal cord injurysite.

In certain embodiments, a cell population in accordance with the presentdisclosure is capable of remaining within the spinal cord injury site ofthe subject for a period of about 180 days or longer followingimplantation of a dose of the composition into the spinal cord injurysite. In other embodiments, a cell population in accordance with thepresent disclosure is capable of remaining within the spinal cord injurysite of the subject for a period of about 2 years or longer followingimplantation of a dose of the composition into the spinal cord injurysite. In further embodiments, a cell population in accordance with thepresent disclosure is capable of remaining within the spinal cord injurysite of the subject for a period of about 3 years or longer followingimplantation of a dose of the composition into the spinal cord injurysite. In yet further embodiments, a cell population in accordance withthe present disclosure is capable of remaining within the spinal cordinjury site of the subject for a period of about 4 years or longerfollowing implantation of a dose of the composition into the spinal cordinjury site.

In certain embodiments, a cell composition in accordance with thepresent disclosure is capable of reducing spinal cord injury-inducedparenchymal cavitation in a subject. In certain embodiments, a lesionvolume is reduced by formation of a tissue matrix in the spinal cordinjury site. In certain embodiments, the cells of the present disclosureare capable of forming a tissue matrix in the spinal cord injury sitewithin about 180 days or less. In certain embodiments, the subject withreduced injury-induced parenchymal cavitation is human.

In certain embodiments, a cell population in accordance with the presentdisclosure can be capable of reducing a volume of an injury-inducedcentral nervous system parenchymal cavitation in about 12 months orless. In certain embodiments, a cell population in accordance with thepresent disclosure can be capable of reducing a volume of aninjury-induced central nervous system parenchymal cavitation in asubject in about 6 months or less, about 5 months or less, or less thanabout 4 months. In certain embodiments, the subject is human.

In an embodiment, one or more cells from a cell population in accordancewith the present disclosure can be capable of migrating from a firstlocation to one or more second locations within the central nervoussystem of a subject in need thereof. In an embodiment, one or more cellsfrom a cell population in accordance with the present disclosure can becapable of migrating from the spinal cord of a subject to an affectedtissue within the brain of the subject. In one embodiment, one or morecells from a cell population in accordance with the present disclosurecan be capable of migrating from a first location within the spinal cordof a subject to a second location at an affected tissue within thespinal cord of the subject. In one embodiment, one or more cells from acell population in accordance with the present disclosure can be capableof migrating from a first location within the brain of a subject to asecond location at an affected tissue within the brain of the subject.In one embodiment, one or more cells from a cell population inaccordance with the present disclosure can be capable of migrating froma first location within the brain of a subject to an affected tissuewithin the spinal cord of the subject. In one embodiment, one or morecells from a cell population in accordance with the present disclosurecan be capable of migrating from a first location within the spinal cordof a subject to a second location at an affected tissue within thespinal cord of the subject, as well as to one or more locations at oneor more affected tissues within the brain of the subject. In oneembodiment, one or more cells from a cell population in accordance withthe present disclosure can be capable of migrating from a first locationwithin the brain of a subject to a second location at an affected tissuewithin the brain of the subject, as well as to one or more locations atone or more affected tissues within the spinal cord of the subject.

In an embodiment, one or more cells from a cell population in accordancewith the present disclosure can be capable of migrating from a firstlocation to one or more second locations at one or more affected tissueswithin the central nervous system of a subject in less than about 150days, such as less than about 100 days, such as less than about 50 days,or such as less than about 10 days. In an embodiment, one or more cellsfrom a cell population in accordance with the present disclosure can becapable of migrating from a first location to one or more secondlocations at one or more affected tissues within the central nervoussystem of a subject in about 180 days or less.

Additional Embodiments

Additional embodiments of the present disclosure include the following:

1. A container containing a composition that comprises a population ofallogeneic human oligodendrocyte progenitor cells (OPCs) that arecapable of engrafting at a spinal cord injury site of a human subjectfollowing implantation of a dose of the composition into the spinal cordinjury site.

2. The container according to 1, wherein the OPCs do not elicit ahumoral or cellular immune response in the human subject within one yearof administration when the subject undergoes a low doseimmunosuppressant regimen that is discontinued at about 60 days afterimplantation of a dose of the composition into the spinal cord injurysite.

3. The container according to either of the embodiments 1 or 2, whereinthe OPCs are capable of remaining within the spinal cord injury site ofsaid subject for a period of about 180 days or more followingimplantation of a dose of the composition into the spinal cord injurysite.

4. The container according to any of the embodiments 1-3, wherein theOPCs are capable of remaining within the spinal cord injury site of saidsubject for a period of about 2 years or more following implantation ofa dose of the composition into the spinal cord injury site.

5. The container according to any of the embodiments 1-4, wherein theOPCs are capable of forming a tissue matrix in the spinal cord injurysite within about 180 days or less, thereby reducing spinal cordinjury-induced parenchymal cavitation.

6. The container according to any of the embodiments 1-5, wherein theOPCs are capable of secreting MCP-1 and one or more factors selectedfrom the group consisting of: Clusterin, ApoE, TIMP1 and TIMP2.

7. The container according to any of the embodiments 1-5, wherein theOPCs are capable of secreting all of the factors MCP-1, Clusterin, ApoE,TIMP1 and TIMP2.

8. The container according to any of the embodiments 1-7, wherein thedose of the composition comprises between about 2×10⁶ and about50×10⁶AST-OPC 1cells.

9. The container according to 8, wherein the dose of the compositioncomprises between about 20×10⁶ and about 50×10⁶ AST-OPC1 cells.

The container according to 8, wherein the dose of the compositioncomprises between about 2×10⁶ and about 20×10⁶ AST-OPC1 cells.

10. A container containing a composition that comprises a population ofallogeneic human oligodendrocyte progenitor cells (OPCs) that arecapable of presenting at a spinal cord injury site of a subject andproduce no detectable systemic toxicity in the subject followingimplantation of a dose of the composition into the spinal cord injurysite.

11. The container according to 10, wherein the OPCs do not inducesignificant alterations in a hematology, coagulation, urinalysis orclinical chemistry parameter of the subject.

12. A method of reducing spinal cord injury-induced parenchymalcavitation in a human subject with an acute spinal cord injury, themethod comprising administering to said subject a composition thatcomprises a population of allogeneic human oligodendrocyte progenitorcells (OPCs) that are capable of engrafting at a spinal cord injurysite.

13. The method according to 12, wherein administering the compositioncomprises directly injecting the composition into the spinal cord injurysite approximately 5 mm caudal of the spinal cord injury epicenter.

14. The method according to either of the embodiments 11-12, furthercomprising administering to the subject a low dose immunosuppressantregimen.

15. The method according to 14, wherein the immunosuppressant regimencomprises a tacrolimus dose of about 0.03 mg/kg/day per os, adjusted tomaintain a trough blood concentration of about 3-7 ng/mL through aboutday 46 following the administering of the composition comprising apopulation of allogeneically derived OPCs, followed by tapering off anddiscontinuing the immunosuppressant regimen at about day 60 followingthe administering of the composition.

16. The method according to 12, wherein the OPCs are capable ofremaining within the spinal cord injury site of said subject for aperiod of about 180 days or longer following the administration of thecomposition to the spinal cord injury site.

17. The method according to 12, wherein the OPCs are capable ofremaining within the spinal cord injury site of said subject for aperiod of about 2 years or longer following the administration of thecomposition to the spinal cord injury site.

18. The method according to any of the embodiments 12-16, wherein theOPCs are capable of forming a tissue matrix in the spinal cord injurysite of said subject within about 180 days or less, thereby reducingspinal cord injury-induced parenchymal cavitation.

19. The method according to any of the embodiments 12-17, wherein thesubject has a thoracic or cervical spinal cord injury.

20. The method according to any of the embodiments 12-19, wherein thecomposition comprises between about 2×10⁶ and about 50×10⁶ AST-OPC1cells.

21. A container containing a composition that comprises a population ofoligodendrocyte progenitor cells (OPCs) that are capable of remainingwithin a spinal cord injury site of a subject at a concentration ofgreater than 1% of all cells present at the spinal cord injury site ofsaid subject for a period of 180 days or more following implantation ofa dose of the composition into the spinal cord injury site of thesubject.

22. The container according to claim 9, wherein the percentage of OPCsthat are present at the spinal cord injury site is measured bequantitative polymerase chain reaction (qPCR).

23. A container containing a composition comprising a population ofoligodendrocyte progenitor cells (OPCs) that are capable of presentingat a spinal cord injury site of a subject and are capable of migratingless than 17 mm within a spinal cord of the subject as measured from amost rostral to a most caudal OPC over a period of about 180 days ormore following implantation of a dose of the composition into the spinalcord injury site.

24. The container according to 23, wherein the OPCs are capable ofmigrating within a white matter tissue or a gray matter tissue of theSpinal Cord.

25. The container according to any one of the embodiments 21-24, whereinthe composition comprises between about 2.0×10⁶ and 50×10⁶ AST-OPC1.

26. A container containing a composition that comprises a population ofOPCs that are capable of presenting at a spinal cord injury site and arecapable of producing no detectable increase in a frequency of allodyniain a subject following implantation of a dose of the composition intothe spinal cord injury site.

27. The container according to 26, wherein the allodynia is measured inresponse to a normally non-noxious blunt force mechanical stimulus.

28. The container according to 26, wherein the allodynia is measured inresponse to a normally non-noxious cold temperature stimulus.

29. The container according to any one of the embodiments 26-28, whereinthe allodynia is measured at two different anatomical sites on thesubject.

30. The container according to any one of the embodiments 26-29, whereinthe OPCs are capable of producing no detectable increase in thefrequency of allodynia for a period of time ranging from 3 to 9 monthspost-implantation.

31. The container according to any one of the embodiments 26-30, whereinthe composition comprises between about 2.0×10⁶ and 50×10⁶ AST-OPC1.

32. The container according to 31, wherein the composition comprises apharmaceutically-acceptable carrier.

33. The container according to either of the embodiments 31 or 32,wherein the composition is formulated for administration via a directinjection into the spinal cord of a subject.

34. The container according to either of the embodiments 31 or 32wherein the composition is formulated for intracerebral,intraventricular, intrathecal, intranasal or intracisternaladministration to a subject

35. A method of inducing myelin repair or remyelination in a subjectcomprising administering a therapeutically effective amount of thecomposition according to any one of the previous claims to the subject.

36. The method according to 35, further comprising treating the subjectto reduce immune rejection of the OPCs.

37. The method according to 36, further comprising administering ananti-inflammatory agent to the subject.

38. The method according to any one of the embodiments 35-37, whereinthe subject has been diagnosed with a disease or pathology selected fromthe group consisting of: multiple sclerosis, leukodystrophy,Guillain-Barre Syndrome, Charcot-Marie-Tooth neuropathy, Tay-Sachsdisease, Niemann-Pick disease, Gaucher disease, and Hurler Syndrome.

39. The method according to any one of the embodiments 35-37, whereinthe subject has been diagnosed with a condition selected from the groupconsisting of: inflammation, stroke, immune disorders, metabolicdisorders, and nutritional deficiencies.

40. The method according to any of the embodiments 35-37, wherein thesubject has suffered a traumatic injury resulting in a loss ofmyelination.

41. The method according to 40, wherein the traumatic injury is an acutespinal cord injury.

42. The method according to 41, wherein the traumatic injury is cervicalspinal cord injury.

43. The method according to 41, wherein the traumatic injury is thoracicspinal cord injury.

42. The method according to any of the embodiments 35-43, wherein thecomposition is surgically administered to the subject.

43. The method according to any of the embodiments 35-43, wherein thecomposition is administered to the subject using a non-invasive deliverymethod.

Materials and Methods

The following paragraphs, describing the materials, systems and methodsof several specific embodiments, are intended to be illustrative onlyand are not to be construed as limiting the scope of the presentdisclosure to the specific features or combinations of featuresdescribed.

Animal Subjects. All procedures used were approved by a board-certifiedveterinarian and were conducted in accordance with the NationalInstitute of Health Guide for the Care and Use of Laboratory Animals.Adult athymic nude rats (strain Crl:NIH-Foxnlrnu-) were obtained fromCharles Rivers Laboratories (Wilmington, Mass.). Rag2^(−/−)γc^(−/−) miceand shiverer^(−/−) mice were obtained from Jackson Laboratories (BarHarbor, Me.) and bred in-house to generate Rag2^(−/−)γc^(−/−)/shi^(−/−)homozygous mice. All animal subjects were housed in standard conditionswith a 12 hr light/dark cycle, were provided food and water ad libitum,and were allowed to acclimate for a minimum of one week prior to surgery

Differentiation of AST-OPC] from hESCs. The WA01 (H1) hESC line wasexpanded in feeder-free conditions (Xu C, Inokuma M S, Denham J, GoldsK, Kundu P, Gold J D, Carpenter M K. Feeder-free growth ofundifferentiated human embryonic stem cells. Nat Biotechnol. 2001October; 19(10):971-74; Li Y, Powell S, Brunette E, Lebkowski J,Mandalam R. Expansion of human embryonic stem cells in definedserum-free medium devoid of animal-derived products. Biotechnol Bioeng.2005 Sep. 20; 91(6):688-98). hESC colonies were lifted with collagenaseand manual scraping and then seeded into ultra-low attachment flasks(Day 0) in 50% hESC growth media and 50% glial progenitor medium (GPM)containing 4 ng/mL of basic fibroblast growth factor (FGF) and 20 ng/mLepidermal growth factor (EGF) to stimulate embryoid body formation. OnDay 1, media was replaced with 50% hESC growth media/50% GPM containing20 ng/mL EGF and 10 μM all-trans-retinoic acid (RA). On Days 2-8, mediawas replaced daily with 100% GPM containing 20 ng/mL EGF and 10 μM RA.On Days 9-26, embryoid bodies were maintained in GPM/EGF media withoutRA, and media was replaced every 2 days. On Day 28, embryoid bodies wereplated in Matrigel-coated flasks and cultured in GPM/EGF media for 7days with media exchange every 2 days. On Day 34, cells were harvestedwith trypsin, replated in Matrigel-coated flasks, and cultured for anadditional 7 days in GPM/EGF media, with media exchange every 2 days. OnDay 41, cells were harvested with trypsin, filtered to remove residualcell aggregates, and cryopreserved in liquid nitrogen.

Analysis of differentiated AST-OPC] by flow cytometry. DifferentiatedAST-OPC1 samples were assayed for the presence of surface andintracellular markers using standard flow cytometry. For surface markerstaining, Day 41 AST-OPC1 samples were blocked with 10% heat-inactivatedgoat serum (HI FBS) and then incubated with primary antibody and/orisotype control (0.5 μg/5×10⁵ cells, NG2, Invitrogen #37-2300; MouseIgG1 BD Biosciences #55412) for 30 minutes at 2-8° C., washed, then andincubated with secondary antibody (goat-anti-mouse-IgG1-A488 InvitrogenA21121 at 0.25 μg/5×10⁵ cells) for 30 minutes at 2-8° C. To excludenonviable cells, propidium iodide (Sigma P4864 at 1 i.t.g/mL) was addedto the stained samples just prior to acquisition. All samples were thenacquired and the data analyzed on the BD Biosciences FACSCalibur™cytometer system using Cellquest Pro software.

For intracellular marker staining, Day 41 AST-OPC1 samples were taggedwith ethidium monoazide (Sigma E2028 at 5 i.t.g/mL) for dead celldiscrimination followed by fixation using 2% Paraformaldehyde (PFA) andthen permeabilization with cold 90% methanol. The cells were blockedwith either 5% HI FBS (for Oct4) or 10% heat-inactivated goat serum (forNestin) and then incubated with primary antibody and/or isotype control(goat anti-Oct4 Santa Cruz SC8629, normal goat IgG SC2028 at 0.15-0.5μg/5×10⁵ cells; Nestin Millipore MAB5326; MoIgG1 BD Biosciences 554121at 0.5 μg/5×10⁵ cells) for 30 minutes at 2-8° C., washed with stainbuffer and incubated with secondary antibody (donkey-anti-goat-IgG-A488Invitrogen A11055 or goat-anti-mouse-IgG1-A488 Invitrogen A21121 at 0.25μg/5×10⁵ cells) for 30 minutes at 2-8° C. All samples were then acquiredand the data analyzed on the BD Biosciences FACSCalibur™ cytometersystem using Cellquest Pro software.

Quantification of secreted factors in AST-OPC] conditioned medium byLuminex. Conditioned media from 7 different AST-OPC1 lots was collectedat the time of harvest (immediately prior to cryopreservation) and sentto AssayGate, Inc. (Ijamsville, Md.) for Luminex-based detection of 66secreted factors. Secreted factors that were detected in all 7 lots andfound to have putative roles in neural repair are reported.

In vitro neurite outgrowth assay. On Day 0, cortical tissue dissectedfrom embryonic day 18 Sprague-Dawley rats was obtained from BrainBits,LLC (Springfield, Ill.) and dissociated to single cells according to themanufacturer's instructions. Dissociated cortical cells were seeded at80,000 cells/0.5 mL/well into poly-L-lysine-coated 24 well plates andcultured in primary cortical neuron medium (PCN medium=Neurobasal Mediumplus 0.5 mM L-glutamine, B-27 supplement, and penicillin/streptomycin,Life Technologies, Grand Island, N.Y.). On Day 4, each well received 0.5mL additional PCN medium and a 0.4 μM pore transwell insert containingeither 200,000 AST-OPC1 cells/0.3 mL PCN medium or 0.3 mL PCN mediumalone. On Days 7 and 10, 50% of the medium in the bottom chamber of eachwell was exchanged.

On Day 14, transwell inserts and PCN medium were removed from each well,and primary cortical neurons were fixed with 4% paraformaldehyde/DPBSfor 15 min at RT, followed by 3 washes in DPBS. Wells were incubated inblocking solution (10% normal goat serum/0.1% Triton X-100/DPBS) for 30min at RT, followed by incubation with the anti-neurofilament antibody,SMI-312 (1:1000, Abcam ab24574) in blocking solution overnight at 4° C.Following 3 washes in DPBS, wells were incubated with a goat anti-mouseIgG-Alexa594 secondary antibody (1:400, Life Technologies A11032) andDAPI in blocking solution for 2 hr at RT. Following 3 washes in DPBS,plates were imaged on an INCell Analyzer 2000 (GE Healthcare,Pittsburgh, Pa.), using a 10× objective to acquire 9 imaging fields perwell. ImageJ software (NIH, Bethesda, Md.) was used to determinefluorescent area measurements of SMI-312 and DAPI positivity in eachimage. Because the SMI-312 antibody exhibited reactivity to cell axonsand nuclei, the total area of DAPI positivity was subtracted from thetotal area of SMI positivity for each image, to determine the total areaof axonal outgrowth. The average axonal outgrowth across the 9 imagingfields was then determined for each well and expressed as a percentagerelative to the overall average axonal outgrowth of vehicle-treatedwells for each culture plate. Results in FIG. 2C are the average ofthree independent experiments.

Thoracic spinal cord injury. Following at least one week of acclimation,athymic nude rats [Crl:NIH-Foxn1^(rnu)] were subjected to a thoracicspinal cord crush/contusion injury at level T10. Rats were given anintraperitoneal (IP) injection of a 60 mg/kg ketamine/7.5 mg/kg xylazinecocktail to induce anesthesia, and then a midline skin incision was madeat the T8 to T11 level of the thoracic Spinal Cord. The paravertebralmuscles were dissected bilaterally to visualize the transverseapophyses. A laminectomy was performed at T10, and a midline contusioninjury was induced using the Infinite Horizons Impactor (PrecisionSystems & Instrumentation, Fairfax, Va.) set to deliver a 200 kdyneforce impact. Following contusion injury and wound closure, animals weregiven a subcutaneous (SC) injection of Lactated Ringer's Solution (LRS;10 mL) and maintained on an isothermic pad until recovery fromanesthesia. Following contusion injury, manual bladder expression wasperformed 2-3 times daily for each animal until voluntary bladderexpression returned.

AST-OPC] transplantation in injured nude rats and Shiverer/Rag2 mice.For athymic nude rats (Crl:NIH-Foxn1^(rnu)), AST-OPC1 was injected intothe spinal cord 6-8 days post-contusion injury. Animals were positionedin a stereotaxic frame, and a 50 μL Hamilton syringe outfitted with a 32gauge needle (1 inch long, 30° beveled tip) was used to deliver vehicle(Hank's Balanced Salt Solution, HBSS) or AST-OPC1 at 2.4×10⁶ or 2.4×10⁵cells/rat into the dorsal spinal parenchyma adjacent to the contusionepicenter via four injections of 64, (high dose AST-OPC1 or HBSS) or asingle 2.4 μL injection (low dose AST-OPC1).

The immunosuppressive article, anti-asialo GM1 antibody (GM1Ab, Wako986-10001) was administered via an IP injection to all athymic nude ratstwo days prior to transplant surgery, on the day of transplantation, andtwo days after transplantation, and weekly thereafter as 1 mg/injectionin 0.2 mL USP sterile saline for injection.

For Rag2^(−/−) γc^(−/−)/shi^(−/−) (Shiverer), AST-OPC1 was injected intothe uninjured spinal cord at T9-T10 at a dose ranging from 2.5×10⁵ to1×10⁶ cells/mouse and at a concentration of 1×10⁵ cells/μL using thesame approach as described for athymic nude rats. No additionalimmunosuppressive agents were given to AST-OPC1-treated mice.

Animal perfusion and tissue processing for histology. Central andperipheral tissues were collected at autopsy and immersion fixed in 10%formalin for paraffin-embedding. For spinal cord, the approximaterostral and caudal extent of the affected spinal cord tissue,corresponding to approximately 1 cm rostral and 1 cm caudal to thecontusion epicenter or site of administration, was dissected en blocduring necropsy. The tissue was processed for paraffin embedding usingstandard procedures. Spinal cord tissue was sectioned in thelongitudinal/horizontal plane by microtome and 5 μm sections wereobtained and mounted onto slides for subsequent hematoxylin and eosin(H&E) staining, ISH and IHC.

Whole body fixation was performed by transcardial perfusion with 0.9%saline followed by 4% PFA. For frozen tissue preparation, perfusedtissue was post-fixed in 4% PFA overnight at 4° C. followed bycryoprotection in 30% sucrose/PBS for 72 hr at 4° C. Cryoprotectedtissue was snap frozen on dry ice and stored at −80° C. until sectioningat 20 μm on a cryostat.

Myelin staining with Eriochrome cyanine. To identify myelinated fiberswithin the spinal cord, tissues were stained with Solochrome cyaninesolution (Fisher Scientific, Pittsburgh, Pa.) for 20 min at RT, rinsedbriefly in tap water, and differentiated in 10% iron alum solution(Fisher Scientific, Pittsburgh, Pa.) for 10 min at RT. Following a briefrinsing in tap water, slides were counterstained with Eosin-Y (BiocareMedical, Concord, Calif.), dehydrated through a sequential ethanolseries, cleared with xylenes and coverslipped with EcoMount (BiocareMedical, Concord, Calif.). Stained slides were imaged with an AxiocamMRc5 camera mounted on an Observer D1 microscope (Carl Zeiss, Gottingen,Germany).

To visualize myelinated fibers, spinal cord tissue sections were washedbriefly in dH2O and incubated for 30 minutes in a 0.2% acidic solutionof Eriochrome (Solochrome) Cyanine RS (EC; Sigma, Cat. #E2502), followedby a 10 min differentiation in 5% iron alum, and several washes in dH₂O.Differentiation was completed with a 10 min incubation inborax-ferricyanide and washes in dH₂O. Sections were dehydrated inascending ethanols, cleared in Histoclear™ and coverslipped withPermount™ (Fisher Scientific).

Characterization of transplanted AST-OPC] by immunohistochemistry & insitu hybridization. All animals that received AST-OPC1 transplantationwere assayed for the presence of human cells in the Spinal Cord. Forparaffin-embedded tissue, in situ hybridization (ISH) was used to labelnuclear human-specific Alu DNA repeat sequences, followed bycolorimetric detection. Briefly, tissues were deparaffinized in xylenesand 100% ethanol and permeabilized using pepsin. Biotinylated Alu probes(Q151P.9900, Invitrogen, Carlsbad, Calif.) were hybridized to the tissuefor 5 min at 95° C. followed by 2 hr at 37° C. Post-hybridizationstringency washes were performed at 37° C. Slides were incubated withhorseradish peroxidase (HRP) conjugate for 30 min at 37° C.Nickel-enhanced diaminobenzidene tetrahydrochloride (Ni-DAB, VectorLaboratories, Burlingame, Calif.) was used for HRP visualization. Slideswere counterstained with nuclear Fast Red, dehydrated through asequential ethanol series, defatted in xylenes and coverslipped withPermount™ (Fisher Scientific, Santa Clara, Calif.).

Immunohistochemistry was performed to identify human cells in fixedfrozen tissue using human nuclei antiserum (MAB1281, Chemicon, 1:500)and to label cells in proliferating phases of the cell cycle (all phasesexcept GO) in paraffin-embedded tissue using Ki67 antiserum (833-500,Abcam, Cambridge, Mass., 1:1000). If necessary, slides weredeparaffinized using xylenes and 100% ethanol. Antigen retrievalpretreatment consisted of placing the slides in boiling citrate buffer(pH 6.0) and microwaving at full power for 10 min, followed by coolingto ambient temperature. Slides were rinsed in DPBS and incubated withblocking buffer (0.3% Triton X-100, 10% normal goat serum, 0.1% bovineserum albumin, 3% H2O2 in DPBS) for 1 hr at ambient temperature followedby incubation with primary antibody for 24 hr at 4° C. The sections werewashed and incubated with biotinylated secondary antibody (BA-1000,Vector Laboratories, Burlingame, Calif., 1:500) for 1 hr at ambienttemperature, washed and incubated with streptavidin-HRP (VectastainElite ABC kit, Vector Laboratories, Burlingame, Calif., 1:1000 in DPBS)for 1 hr at ambient temperature. Ni-DAB, (Vector Laboratories) was usedfor HRP visualization. Slides were counterstained with eosin, dehydratedthrough a sequential ethanol series, defatted in xylenes andcoverslipped with Permount™ (Fisher Scientific, Santa Clara, Calif.).

Cavitation area measurements in the injured Spinal Cord. Measurements ofcavity formation were performed on spinal cord tissues sectioned in thelongitudinal (horizontal) plane and stained with Eriochromecyanine/Eosin-Y as described above. Measurements were made on spinalcord sections from 10-12 subjects in each treatment group (2.4×10⁵AST-OPC1, 2.4×10⁶ AST-OPC1 or HBSS), using a single tissue section thatcontained the injury/graft epicenter for each animal. Area measurementswere performed using ImageJ software (NIH, Bethesda, Md.) withoutknowledge of treatment group.

Biodistribution of transplanted AST-OPC] by quantitative PCR. One weekafter thoracic spinal cord injury, rats were transplanted with twodifferent doses of AST-OPC1 (2.4×10⁵ or 2.4×10⁶ AST-OPC1) or HBSSvehicle control, using the methods described above. At 2, 14, and 180days post-administration, blood, cerebral spinal fluid (CSF) and tissueswere collected from the transplanted rats. These time points were chosento reflect times before and after the expected restoration of thefunctional blood-spinal cord barrier, after which AST-OPC1 was unlikelyto migrate out of the central nervous system.

One set of animals at each time point had samples harvested andprocessed for quantitative PCR (qPCR). At 2, 14 and 180 days afterAST-OPC1 transplantation, animals were euthanized via CO₂ asphyxiationand subsequent exsanguinations and the following tissues were collected:spinal cord (cervical cord/brainstem, lumbar/thoracic cord), brain(cerebellum, forebrain), lungs, heart, liver, spleen, CSF, blood,meninges, kidney, small intestine and ovaries/testes. Tissues and CSFwere flash frozen in isopentane chilled on dry ice and stored at 80° C.The presence of human DNA was assayed by amplifying a 232 base pairsequence of the human Alu Y repeat sequence using the ABI Prism 7700Sequence Detection System. The mass of human genomic DNA detected in onemicrogram of rat genomic DNA extracted from each tissue was quantifiedusing serial dilutions of human genomic DNA as standards. The lowerlimit of detection in the assay used was 100 fg human genomic DNA/μg ratgenomic DNA. A positive signal for these sequences was interpreted asthe presence of human cells in the tissue from which the DNA wasextracted. All qPCR analyses were performed by Althea Technologies (SanDiego, Calif.).

Also at 2, 14, and 180 days after AST-OPC1 transplantation, parallelsets of animals in each group were transcardially perfused with ice-cold0.9% saline followed by ice-cold 4% paraformaldehyde in Sorensen'sphosphate buffer. Spinal cords and brains were dissected and post fixedin 4% PFA overnight at 4° C. and cryoprotected with 30% sucrose inDulbecco's phosphate buffered saline (DPBS) at 4° C. Tissue was embeddedin Tissue-Tek O.C.T. compound (Ted Pella, Redding, Calif.) and frozen ondry ice. Spinal cords were sectioned longitudinally through the site ofthe cell or vehicle injections, extending approximately 2 cm in bothrostral and caudal directions from the contusion epicenter. Brains weresectioned in the coronal plane at the levels of the olfactory bulbs,lateral ventricles, hippocampus and cerebellum. 20 μm cryosections werethaw-mounted onto SuperFrost Plus™ slides (Fisher Scientific, Fair Lawn,N.J.) and stored desiccated at −80° C. until use.

Clinical and toxicological assessments of AST-OPC1-treated, contusedrats. Toxicology studies of AST-OPC1-treated, contused male and femaleathymic nude rats were performed at MPI Research (Mattawan, Mich.) underGLP conditions. Observations for morbidity, mortality, injury, and theavailability of food and water were conducted at least twice daily forall animals enrolled in toxicology studies. Clinical observations wereconducted and body weights were measured and recorded twice weeklyduring the study. At study termination, necropsy examinations wereperformed, organ weights were recorded, blood and urine samples werecollected for clinical pathology evaluations and selected tissues wereexamined microscopically by a board-certified veterinary pathologist whowas blinded to animals' treatment groups.

Allodynia measurements on AST-OP Cl-treated, contused rats. Atapproximately 3, 6, and 9 months post-transplantation, 10 male and 10female rats were evaluated for allodynia or hypersensitivity in responseto normally non-noxious mechanical (blunt probe) or cold (pointapplication of acetone) stimuli, as described previously (Hulsebosch CE, Xu G, Perez-Polo J R, Westlund J R, Westlund K N, Taylor C P, McAdooD J 2000 Rodent model of chronic pain after spinal cord contusion injuryand effects of gabapentin. J. Neurotrauma 17(12):1205-1217). Baselinemeasurements were performed on uninjured non-transplanted rats (18male/20 female). For each animal tested, stimuli were applied to theanimal's dorsum for 3 trials at each point of a 9 point grid centered onthe laminectomy site (27 mechanical stimuli and 27 cold stimuli per timepoint) and stimuli were applied to the glabrous surface of each paw for5 trials of each stimulus (20 mechanical stimuli and 20 cold stimuli pertime point). Each animal tested was observed for supraspinal responsesto the stimuli according to the following parameters: three anatomicallevels of assessment (at, above, and below the level of injury), twomodalities (application of mechanical stimulation or cold stimulation),and two assay sites (dorsal skin surface of the trunk and glabroustissue of the paws). The mechanical and cold hypersensitivity tests onthe dorsal skin were separated by at least 2 hours for each animal. Thedorsal skin tests were conducted on freely moving, non-anesthetizedanimals in a home cage or familiar environment. Testing on the glabroustissue was conducted in a Plexiglas™ box test apparatus with wire meshfloor. Animals were habituated to the apparatus for 10 min prior totesting. With each modality, animals were randomly assigned to receivemechanical or cold stimuli first.

Mouse Tumorigenicity Studies of AST-OPC] spiked with undifferentiatedhESCs. Tumorigenicity studies were performed at MPI Research (Mattawan,Mich.) under GLP conditions. AST-OPC1 spiked with its parent hESC line,H1, was administered into the thoracic spinal cord of uninjured male andfemale CB-17/IcrCrl-Prkdc^(scid)Lyst^(bg)BR mice using thetransplantation procedure described above. Animals were administered atotal dose of 2×10⁶ cells or HBSS as a single 0.01 mL injection and weremonitored for up to 12 months for clinical signs of tumor formation:Cell-treatment groups and sample sizes were as follows: 100% H1, n=30;50% H1, n=31; 10% H1, n=31; 5% H1, n=12; 1% H1, n=37; 100% AST-OPC1,n=129; HBSS, n=30. Microscopic examination of fixed hematoxylin andeosin (H&E) stained paraffin sections was performed onprotocol-designated sections of tissues. The slides were examined by aboard certified veterinary pathologist. A four-step grading system wasutilized to define gradable lesions for comparison between treatmentgroups. Presence of human cells in observed tumors was confirmed by ISHas described above.

EXAMPLES

The following examples are not intended to limit the scope of what theinventors regard as their invention nor are they intended to representthat the experiments below are all or the only experiments performed.

Example 1: Derivation and Characterization of AST-OPC1

AST-OPC1 (formerly known as GRNOPC1) was generated by thedifferentiation of WA01 (H1) hESCs from a master cell bank (MCB) asdescribed in the Materials and Methods. The differentiation process toproduce AST-OPC1 requires 41 days and transitions the hESCs fromundifferentiated cell colonies through embryoid bodies to become anadherent, dispersed cell population which is harvested andcryopreserved. Representative photomicrographs of the cells at differentstages of the 41 day differentiation process are shown in FIG. 1A.

Analysis of 41 day differentiated AST-OPC1 by flow cytometry andimmunocytochemistry (ICC) indicated that the cell population wascomprised mostly of neural lineage cells of the early oligodendrocyteprogenitor phenotype. By flow cytometry, over 90% of the cells werepositive for Nestin and >50% were positive for NG2, a neural/glialproteoglycan expressed by oligodendrocyte progenitor cells (FIG. 1B). Inaddition, levels of the pluripotent stem cell marker, Oct4, were belowthe level of quantitation (<0.2%), indicating a lack of residual hESCs(FIG. 1B). Using an alternative high content image analysis assay, wefurther determined that the frequency of Oct4+ cells in AST-OPC1 wasless than 0.05% (data not shown). Using the defined AST-OPC1differentiation process, we produced over 75 lots of AST-OPC1, which wefurther characterized by ICC on day 41 for the presence of multiplemarkers of ectodermal, mesodermal, endodermal, and pluripotent celltypes to assess the composition of the population and detect potentialunwanted cells types (FIG. 9). In agreement with the flow cytometryresults, ICC profiling indicated a cell population predominantlycomposed of early oligodendrocyte progenitor cells with few matureneuronal or astrocytic cells. The presence of endodermal, mesodermal orpluripotent cell types was undetectable to <1% of the differentiatedAST-OPC1 cell population.

Example 2: Stimulation of Axonal Outgrowth In Vitro and Myelination InVivo by AST-OPC1

A non-contact co-culture system using rat primary cortical neurons wasused to assess the ability of AST-OPC1 to induce axonal outgrowth invitro via paracrine signaling. Rat primary cortical neurons that werecultured 14 days in control medium (FIG. 2A) or with AST-OPC1 (FIG. 2B)and labeled by ICC with antibodies against the axonal marker SMI-312.Quantitative enumeration of the area of axonal outgrowth from 3independent experiments demonstrated significantly greater axonaloutgrowth with AST-OPC1 co-culture (FIG. 2C).

To assess the ability of AST-OPC1 to induce myelination of axons,cryopreserved AST-OPC1 cells were thawed and injected into the spinalcord of immunodeficient Rag2^(−/−)γc^(−/−)/shi^(−/−) (Shiverer) micethat displayed a dysmyelinated phenotype due to their deficiency inmyelin basic protein production. Two doses of AST-OPC1 (2.5×10⁵ or 1×10⁶cells) were injected into the uninjured spinal cord at thoracic levelT9-T10. The mice were assessed two months post-implant for the presenceof human cells and myelinated axons. Two months after implantation,human cells were detected by immunohistochemistry (IHC) using a humannuclear antigen antibody (hNUC, brown) in close association withmyelinated fibers labeled with Eriochrome cyanine (EC, blue) within thethoracic spinal cord (FIG. 2D). No myelinated fibers or human cells wereobserved outside the vicinity of the graft site (FIG. 2E).

AST-OPC1 survived in the lesion site of rats with spinal cord injuriesand preserved myelinated fibers and reduced cavity formation afterthoracic spinal cord injury. In a collection of studies, athymic nuderats [Crl:NIH-Foxnlrnu] received a 200 kdyne contusion injury at T10using the Infinite Horizons Impactor (Precision Systems &Instrumentation, Fairfax, Va.) and were implanted with 2.4×10⁵ or2.4×10⁶ AST-OPC1 or HBSS vehicle control at 6-9 days post-injury asdescribed in the Materials and Methods. Nine months post-implantation,the lesion sites were examined histologically. In control injured ratsinjected with vehicle, extensive parenchymal cavitation was observed(FIGS. 3A and 3C). The cavity was often large, extending rostrocaudallyacross multiple spinal cord segments from the epicenter of the injurysite. Little to no cellularity existed in the cavity. Myelinated axonsapproached the injury cavity and halted their progression as evidencedby the presence of dystrophic endbulbs, or changed path to circumventthe contusion cavity. By contrast, injured animals injected withAST-OPC1 showed much less cavitation (FIGS. 3B and 3D). In sections fromthe AST-OPC1 transplanted animals, myelinated axons stained with ECcould be seen to enter and traverse the injury site. Similar resultswere observed in 7 individual studies where AST-OPC1 was injected intonude rats with thoracic contusion injuries. In the largest study,parenchymal cavitation was quantified for a subset of animals by ahistologist who was blinded to the animals' treatment group. In controlanimals injected with HBSS vehicle, measurable cavitation at the injurysite was common and observed in 11 out of 12 (92%) animals, whichtypically exhibited large cavities ranging from 120-4520 mm² (FIG. 3E).By contrast, rats injected with AST-OPC1, regardless of dose, showedreduced parenchymal cavitation with 12 out of 21 animals showing noinjury-related cavities (low dose AST-OPC1 animals with cavitation,range=120-1985 mm²; high dose AST-OPC1 animals with cavitation,range=20-1185 mm², FIG. 3E). A statistically significant reduction inmean cavitation area was observed with the high dose of AST-OPC1(p=0.0325), while a similar, non-significant trend was observed with thelow dose of AST-OPC1 (p=0.0814). Within the lesion site of AST-OPC1treated animals, positive labeling with a human-specific Alu DNA repeatsequence probe (hAlu) using in situ hybridization (ISH) confirmed thathuman cells were resident in the lesion site in the area of themyelinated fibers at 9 months post-implant (FIG. 3F).

Example 3: Identification and Quantification of Factors Secreted byAST-OPC1

To identify candidate paracrine factors mediating AST-OPC1 biologicalactivity, including stimulation of axonal outgrowth in vitro andmyelination in vivo, conditioned media from 7 different AST-OPC1 lotswas collected at the time of harvest (immediately prior tocrypreservation) and sent to Assaygate, Inc. (Ijamsville, Md.), forLuminex® assay for detection and quantification of 66 secreted factors[AST-OPC1-conditioned medium contained multiple proteins with putativeroles in neural repair. Consistent with previous findings (Zhang Y W,Denham J, Thies R S. Oligodendrocyte progenitor cells derived from humanembryonic stem cells express neurotrophic factors. Stem Cells Dev. 2006December; 15(6):943-52), all 7 tested AST-OPC1 lots secreted MCP-1, afactor thought to act as a chemoattractant for neural precursor cells(Tang S K, Knobloch R A, Maucksch C, Connor B. Redirection ofdoublecortin-positive cell migration by over-expression of thechemokines MCP-1, M1P-1α and GRO-α in the adult rat brain. Neuroscience.2014 February: 240-248) and promoting glial differentiation of neuralprecursor cells (Gordon R J, Mehrabi N F, Maucksch C, Connor B.Chemokines influence the migration and fate of neural precursor cellsfrom the young adult and middle-aged rat subventricular zone. ExpNeurol. 2012 January; 233(1):587-94). It was discovered that all 7tested lots further secreted the following factors: Clusterin, ApoE,TIMP1 and TIMP2 (FIG. 10).

TABLE 1 Quantification of secreted factor production in individualAST-OPC1 lots. Secreted Factor OPC1 OPC1 OPC1 OPC1 OPC1 OPC1 OPC1(pg/mL) Lot A Lot B Lot C Lot D Lot E Lot F Lot G Clusterin 44,63945,924 11,255 20,563 13,355 13,716 59,170 MCP1 10,118 10,086 10,04810,040 10,039 10,136 10,037 APOE 2,558 3,637 1,330 2,694 968 2,995 3,904TIMP1 2,580 4,149 1,863 1,263 1,064 2,778 6,150 TIMP2 2,064 2,165 9161,008 733 1,084 2,064

Conditioned medium sampling was performed on the final day of AST-OPC1production, prior to cell cryopreservation. Conditioned medium wasassayed for the presence of 66 secreted factors using a standardLuminex® platform. Concentrations are shown as the mean and standarddeviation of the 7 assayed lots. Abbreviations: ApoE, apolipoprotein E;MCP-1, monocyte chemoattractant protein 1; TIMP1 & TIMP2, tissueinhibitor of metalloproteinases 1 and 2. Further details on thecollection of AST-OPC1 conditioned medium and secreted factor detectionby Luminex® are reported in Priest Calif., Manley N. C., Denham J, WirthE D 3rd, Lebkowski J S, Preclinical safety of human embryonic stemcell-derived oligodendrocyte progenitors supporting clinical trials inspinal cord injury, Regen Med. 2015 November; 10(8):939-58.

Example 4: Biodistribution of AST-OPC1

We conducted a biodistribution study to examine the potential ofAST-OPC1 to migrate within the spinal cord and distribute to tissuesoutside the spinal cord following direct administration into the injuredthoracic Spinal Cord. One of two doses of cryopreserved AST-OPC1(2.4×10⁵ or 2.4×10⁶) was administered into athymic nude rats 6-9 daysafter a 200 kdyne T9-10 spinal cord contusion injury and animals weremaintained for 2, 14 and 180 days to examine biodistribution of thecells. These time points were chosen to reflect times before and afterthe expected restoration of the functional blood-spinal cord barrier,after which AST-OPC1 was unlikely to migrate out of the central nervoussystem. Ten females were included at each of the three time points foreach of the three treatment groups (HBSS vehicle control, 2.4×10⁵AST-OPC1 or 2.4×10⁶ AST-OPC1). For the 180 day time point, an additionalgroup of 10 males was included which received the high dose of AST-OPC1.At 2, 14 and 180 days post-administration, 5 animals from each timepoint and treatment group were euthanized and had central nervous systemand peripheral tissues collected including spinal cord (cervicalcord/brainstem, lumbar/thoracic cord), brain (cerebellum, forebrain),blood, gonads, liver, heart, lung, kidney, spleen and small intestine.Samples were homogenized and assayed for the presence of human cellsbased on detection of hAlu DNA by quantitative polymerase chain reaction(qPCR). The lower limits of quantitation and detection for hAlu by qPCRin this assay was 1 pg human genomic DNA/μg rat DNA and 100 fg humangenomic DNA/μg rat DNA, respectively.

At all time points assayed, hAlu was detected at less than the limit ofquantification (<1 pg human gDNA/μg nude rat gDNA) in all rat peripheraltissues sampled, including blood, gonads, liver, heart, lung, kidney,spleen, and small intestine. Of the 280 peripheral tissue samplesanalyzed for hAlu by qPCR, only 9 (3.2%) showed detectable butnon-quantifiable levels of human DNA. This frequency was close to theincidence of false positive samples (2 of 120 or 1.7%) observed intissues from animals injected with HBSS. These data thus suggested that,if present, human cells were very rare in peripheral tissues.

At all time points, AST-OPC1 was found in the CNS, especially at thelesion site of the thoracic spinal cord (FIG. 4A). In most cases, hAluwas detected in the thoracic spinal cord at >10,000 pg human DNA per μgrat DNA (>1.0% human cells). The amount of human DNA in the thoracicspinal cord increased between 2 days and 180 days, although thedifference in human DNA in the thoracic spinal cord did not appear toincrease linearly with the dose of AST-OPC1.

In the cervical spinal cord, most animals had much reduced butmeasurable hAlu levels (>1-10,000 pg human DNA/μg nude rat DNA). Onlytwo animals (1 at the 180 day time point and 1 at the 2 day time point)that received 2.4×10⁶ AST-OPC1 had high levels of hAlu in this region(>10,000 pg human DNA/μg nude rat DNA).

Only 1 rat in the 180 day termination group receiving 2.4×10⁶ AST-OPC1had high levels of hAlu (>10,000 pg human gDNA/ug nude rat gDNA) in thecerebellum. However, this data point may reflect a spurious result, asvery few human cells were detected in the animal's adjacent cervicalspinal cord, a site more proximal to the injection site. An additional 3animals in the 180 day termination group had low levels of hAlu in thecerebellum (>1-100 pg human DNA/μg nude rat DNA).

These PCR-based biodistribution results were confirmed by histologicalanalysis of serial tissue sections of the thoracic spinal cord extendingfrom caudal of the injury site, through the injury site, and rostral tothe cerebellum from the remaining 5 animals per treatment group at eachtime point. The greatest concentration of human cells as identified byIHC for hNUC was at the injection site in area of the injury (FIG. 4B).The concentration of parenchymal AST-OPC1 diminished with distance fromthe injury epicenter. AxioVision image analysis software (AxioVS 40 V4.6.3.0, Carl Zeiss Imaging Solutions) was used to measure the maximumrostrocaudal length of spinal cord between hNUC positive cells. Therostrocaudal extent of AST-OPC1 intraparenchymal distribution increasedwith time post-transplant, with distances of 15-17 mm observed betweenthe most rostral and caudal migrating cells at the 180 day time point.AST-OPC1 migrated within the gray and white matter of the parenchyma anddid not appear isolated by glial scarring or other anatomical barriers.Further analysis showed that, while greater numbers of AST-OPC1 migratedaway from the injury epicenter in the animals that received the highdose of cells, the extent of migration was independent of the cell doseadministered in this study.

Example 5: Toxicology Assessment of AST-OPC1

Three toxicology studies were conducted in accordance with GoodLaboratory Practices (GLP) which investigated the potential toxicity ofAST-OPC1 for the treatment of spinal cord injury. These studiesspecifically addressed potential toxicity issues of AST-OPC1 related toits 1) delivery to the spinal cord, 2) impact on organ function, 3)induction of allodynia, and 4) tumorigenicity. For the toxicologystudies, a rat spinal cord contusion injury was utilized to mimic asclosely as possible the conditions that will be encountered withpatients with non-lacerating spinal cord crush injuries. In each study,rats were given a moderate 200 kdyne contusion injury and transplantedwith either cryopreserved AST-OPC1 or HBSS vehicle. For transplantation,cryopreserved, thawed and prepared AST-OPC1 was injected approximately6-9 days post-injury into the lesion site of these animals using thecell delivery methodology intended for use in the proposed clinicaltrial. The three toxicology studies examined contused male and femalerats, of which 299 and 285 were injected with vehicle and AST-OPC1,respectively. AST-OPC1 doses of 2.4×10⁵ and 2.4×10⁶ cells were assessed.

Vehicle and AST-OPC1 transplanted rats were examined at 2, 6, 9 and 12months post-injury for any negative impact on systemic organ function asmeasured by testing blood and urine analytes. FIG. 11 describes themetabolic and hematologic parameters examined to assess the potentialsystemic toxicity of AST-OPC1. The collective data from these studiessuggested that AST-OPC1 did not induce any significant alterations inhematology, coagulation, urinalysis or clinical chemistry parameterscompared to injured vehicle control animals. Some elevation inindividual parameters, especially in liver enzymes and urea nitrogenwere observed occasionally in animals receiving either AST-OPC1 orvehicle, likely due to the spinal cord injury itself or to the prolongeduse of immunosuppression. There were no statistically significantdifferences in the mortality, body weights or clinical observationsincluding behavioral activity, excretion, external appearance, or skincondition between the AST-OPC1 and vehicle treated contused rats. Acommon cause of death in all groups was septicemia/inflammation, notrelated to AST-OPC1 but likely a reflection of the immunocompromisedstate of the animals and their spinal cord injury. Many deaths were alsorelated to urogenital dysfunction, inflammation, obstruction and/orcalculi. The occurrence or frequency of these urogenital events was notconsidered related to AST-OPC1, as they were observed with similarfrequency across all groups.

Many individuals with spinal cord injuries report musculoskeletal,neuropathic and visceral pain (Siddall P J, Taylor D A, McClelland J M,Rutkowski S B, Cousins M J. Pain report and the relationship of pain tophysical factors in the first 6 months following spinal cord injury.Pain. 1999 May; 81(1-2):187-97; Siddall P J, McClelland J M, Rutkowski SB, Cousins M J. A longitudinal study of the prevalence andcharacteristics of pain in the first 5 years following spinal cordinjury. Pain. 2003 June; 103(3):249-57). To investigate this, allanimals that received spinal cord injuries were monitored forautophagia, and other general behavioral indicators of pain. Across thethree toxicology studies, only 7 of 584 injured rats (3 vehicle and 4AST-OPC1) showed signs of autophagia.

Additional behavioral tests were performed to assess if AST-OPC1impacted the frequency of allodynia in rats with spinal cord injuries.At approximately 3, 6, and 9 months post transplantation, animals in thelargest toxicology study were evaluated for allodynia, hypersensitivityin response to normally non-noxious mechanical (blunt probe) or cold(point application of acetone) stimuli. To assure that the mechanicalprobe and cold (acetone) stimuli were non-aversive, the methods wereinitially used on the dorsal skin surface of 18 random male and 20random female healthy rats from the study prior to laminectomy toestablish baseline responses. Each animal tested was observed forsupraspinal responses to the stimuli according to the followingparameters: three anatomical levels of assessment (at, above, and belowthe level of injury), two modalities (application of mechanicalstimulation or cold stimulation), and two assay sites (dorsal skinsurface of the trunk and glabrous tissue of the paws). At 9 monthspost-administration, animals treated with AST-OPC1 did not showsignificant changes in their response profile, relative to vehiclecontrols, for either modality or at any anatomical sites, and did notdisplay signs of allodynia (FIG. 5). Data are expressed as percentpositive responses.

Example 6: Tumorigenicity Assessment of AST-OPC1

Macroscopic and microscopic examinations were performed by anindependent veterinary pathologist who was blinded to the animals'treatment groups to ascertain whether AST-OPC1 administration resultedin any particular pathology either within or outside the central nervoussystem. Such analysis also included assessments for teratoma or ectopictissue formation. For this analysis, teratomas were defined as expansileproliferations or masses which appeared to have arisen from at least 2different embryonic germ layers (endodermal, mesodermal and/orectodermal). Ectopic tissue was defined as tissue not normally occurringin the tissue or organ examined. For these analyses over 50 tissues wereexamined from each animal including the longitudinal extent of thespinal cord and 5 levels of the brain. There were no macroscopic ormicroscopic pathologic findings outside the spinal cord that wereassociated with AST-OPC1 in either male or female rats from the 2, 6, or9 month termination groups.

The entire length of the thoracic and cervical spinal cords and 5 levelsof the brain (medulla/pons, cerebellum, midbrain, forebrain andolfactory bulbs) were examined for teratomas and ectopic tissue.Teratomas were not observed in the spinal cord or brain in any of the252 animals examined 2, 6, or 9 months post-administration. In agreementwith this, cells within the injury/graft site exhibited very lowpositivity for the proliferation marker, Ki67, at 9 months postadministration. Further, ISH labelling with an hAlu probe indicatedrobust graft survival in the thoracic spinal cord of 239 out of the 252assessed animals examined 2, 6, or 9 months post-administration.

In 6 of the 252 animals injected with AST-OPC1, small “cystic-likeepithelial structures” were observed. Of the 6 observed instances ofcyst formation, 2 were observed in animals that received a dose of2.4×10⁵ AST-OPC1 and 4 were observed in animals that received a dose of2.4×10⁶ AST-OPC1. These cystic structures were confined to the lesionsite, ranged in size from 34-980 μm in diameter and were smaller thantypical parenchymal cavitation seen in vehicle controls. In 3 of these 6animals, 2-3 separate cystic structures were observed in close proximityto one another in the histological sections and may have representedlobules of a single structure. A thoracic spinal cord tissue sectioncontaining one of these cystic structures is shown in FIG. 6A. Cysticstructures were lined with cells exhibiting epithelial morphology (FIG.6B), were of human origin (FIG. 6C), and were not highly proliferativegiven that very few cells were positive for the proliferative marker,Ki67 (FIG. 6D). Cystic structures were never observed in healthy tissueor in the cervical spinal cord or brain of any animal in the study.There were no apparent clinical symptoms in the animals in which cysticstructures were observed.

Example 7: Additional Teratoma Assessments of AST-OPC1

The potential for teratoma formation by AST-OPC1 was also tested in thespinal cord of CB-171IcrCrl-Prkdc^(scid)Lyst^(bg)BR (SCID/bg) mice at 12months post-administration for two different lots of AST-OPC1. In thesestudies, 2×10⁶ AST-OPC1 were administered to the intact, uninjured T10thoracic spinal cord of the immunocompromised mice. A positive controlgroup received undifferentiated hESCs, and a negative control groupreceived HBSS vehicle alone. Additional treatment groups received atotal of 2×10⁶ cells containing AST-OPC1 spiked with 1, 5, 10, or 50%undifferentiated hESCs. Treatment groups consisted of equal number ofmales and females and were followed for 12 months to assess tissues forteratoma or any other tumor formation. Animals euthanized prior to 12month follow-up were also examined at termination for teratomaformation. ISH with hAlu was performed for all recovered spinal cordsand confirmed that human AST-OPC1 persisted in >97% of animals.

No teratomas were observed in mice that were injected in the uninjuredspinal cord with vehicle alone. Nine of 20 (45%) of these vehiclecontrol animals survived to the 12 month termination point. By contrast,teratomas were observed in the majority of animals injected with 100%undifferentiated hESCs. In these animals, 20 of the 30 (67%) miceinjected with 2×10⁶ undifferentiated hESCs developed teratomas in thespinal cord (FIG. 7). Only 6 of the 30 (20%) animals survived to the 12month termination point. None of the 128 mice injected with 2×10⁶AST-OPC1 and examined histologically showed any evidence of teratomasand 51% survived throughout the 12 month in life phase. In addition,AST-OPC1 spiked with 50% undifferentiated hESCs produced teratomas inapproximately 70% of mice. With lower numbers of undifferentiated hESCs,the frequency of teratoma formation decreased. Only 13% (4 of 31) micereceiving AST-OPC1 spiked with 10% hESCs produced teratomas, whereasAST-OPC1 spiked with 5% and 1% undifferentiated hESC did not lead toteratoma or tumor formation (0 of 12 and 0 of 36, respectively).

Example 8: Phase I Thoracic Spinal Cord Injury Clinical Trial ofAST-OPC1 Cells

Subjects with neurologically complete, subacute spinal cord injuries(SCI) were recruited in an open-label, multicenter safety study ofAST-OPC1 cells. The main inclusion criteria were a traumatic,non-penetrating SCI with single neurological level from T3-T10.Potential subjects had to provide informed consent from 3 to 11 daysafter their injury so that AST-OPC1 could be administered 7-14 daysafter SCI. In addition, prospective participants were required toprovide consent for both a primary protocol, under which they werefollowed for one year, and for a long-term follow-up protocol, underwhich they will be followed for an additional 14 years.

Five eligible subjects received a single dose of 2×10⁶AST-OPC1 cells ina dedicated surgical procedure for the study. The cells wereadministered via direct injection to the spinal cord approximately 5 mmcaudal to the lesion epicenter using a syringe positioning devicespecifically designed for this purpose. Subjects received a low dose oftacrolimus (initially 0.03 mg/kg/day PO, then adjusted to maintain atrough blood level of 3-7 ng/mL) which was tapered at day 46 anddiscontinued at day 60. The primary endpoint of the study was safetywith the secondary endpoint being neurological function. Safety wasassessed with respect to AST-OPC1 itself, the procedure to deliver thecell product and the transient immunosuppression used subsequent toimplantation. All five subjects who received AST-OPC1 in the primaryprotocol were enrolled in the long-term follow-up protocol.

The study incorporated multiple safety assessments and proceduresincluding: frequent neurological exams, frequent standardized ISNCSCI(International Standards for Neurological Classification of Spinal CordInjury) exams, serial MM scans of the spine and brain, stopping rulesfor precertified safety concerns, and real time notification of anindependent Data Monitoring Committee (DMC) for pre-specified safetyconcerns.

Safety results. Both AST-OPC12 cells and the immunosuppression regimewere well tolerated. There were no serious adverse events (SAEs)associated with AST-OPC1 either during the primary protocol or duringthe long-term follow-up. A total of three SAEs (pyelonephritis, Grade 2;urinary tract infection, Grade 3; and autonomic dysreflexia/dyspnea,Grade 3) were reported, none of which were considered to be associatedwith AST-OPC1, injection procedure or immunosuppression. Adverse eventspossibly associated with AST-OPC1 included transient low grade fever (1incident) and neuralgia/burning sensation in trunk and lower extremities(4 incidents in one subject). Additionally, there were 16 incidents ofgrade 1 or 2 adverse events possibly associated with immunosuppression,including nausea, urinary tract infection and low magnesium bloodlevels.

Magnetic resonance imaging (MRI) data. Serial MRI scans of the entirespinal cord (visualized on scans of the cervical and thoracic spine)were obtained at screening and at 7, 30, 90, 120, 180, 270 and 365 daysafter AST-OPC1 injection. MRI scans of the brain were also obtained atscreening and at selected postoperative time points. The subjectscontinued (and still continue) to have annual MRI scans of the brain andspinal cord under the long-term follow-up protocol. Under the primaryprotocol, no subject exhibited evidence of an enlarging cyst, enlargingmass, spinal cord damage related to the injection procedure,intramedullary hemorrhage, cerebrospinal fluid (CSF) leak, epiduralabscess or infection, inflammatory lesions of the spinal cord, CSF flowobstruction, or masses in the ventricular system.

In addition to the MRI readouts focused on potential safety relatedissues (described above), an additional review of the scans wasconducted to assess lesion site parenchymal cavitation and the potentialactivity of AST-OPC1 cells. Potential activity of AST-OPC1 cells may bedemonstrated by the presence of tissue in place of the lesion cavitiesin study subjects.

The lesion site parenchymal cavitation review focused on the MRI axialand sagittal T1, T2, and STIR sequences for each subject. Time pointsincluded the baseline MRI scans, selected scans during the first year offollow up, the scan at day 365 (i.e. Year 1) visit, and all subsequentscans during the long-term follow-up to date.

Significant variability in lesion severity and morphology betweensubjects was noted on MRI, despite the fact that clinically all subjectswere American Spinal Injury Association (ASIA) Impairment scale Grade A.In all subjects, the rostral and caudal lesion margins were sharplydemarcated on sagittal images by Day 90 and were stable thereafter. In 4of 5 subjects, the lesion/graft sites were hyperintense on theT2-weighed images at later time points (commencing at Day 180 and up toYear 4, which is the latest time point data currently available for 4out of 5 subjects), with the signal intensity less than that ofcerebrospinal fluid (FIG. 12A and FIG. 12B). This is consistent with thepresence of viable graft tissue in the lesion site (Wirth, E D 3^(rd),Theele D P, Mareci T H, Anderson D K, Reier P J. Dynamic assessment ofintraspinal neural graft survival using magnetic resonance imaging. ExpNeurol. 1995 November: 136(1): 64-72), although it is possible thatgraft tissue could be mixed with some residual host tissue, scar tissue,and/or microcysts. The lesion/graft site signal intensity was similar tocerebrospinal fluid in the remaining subject, suggesting lack of viablegraft tissue in the lesion site. Overall, these data support preventionor reduction of lesion cavity formation in four of the five subjectsduring both the one year primary protocol and the long-term follow-upprotocol. Parenchymal cavitation may have been substantially preventedor reduced by the formation of a tissue matrix in the lesion site.

TABLE 2 Summary of spinal cord MRI findings during initial and long-termfollow-up of five subjects with neurologically complete, subacutethoracic SCI that received an injection of 2 × 10⁶ AS T-OPC1 cells. MRIscan date T2 sagittal T2 axial Day 30 5/5 spinal cord edema 5/5 spinalcord edema consistent with subacute SCI consistent with subacute SCI 5/5no adverse findings as a 5/5 no adverse findings as a result of OPC1injection result of OPC1 injection Day 90 4/5—edema gone or nearly4/5—edema gone or nearly gone with no evidence of a gone with noevidence of a cystic lesion cavity forming cystic lesion cavity forming1/5—lesion is uniformly 1/5—lesion is uniformly bright with intensitysimilar bright with intensity similar to cerebrospinal fluid (CSF) toCSF Day 180 4/5 subjects—hyperintense, 4/5 subjects—hyperintense,intensity less than that of intensity less than that of CSF (consistentwith CSF (consistent with presence of viable graft tissue presence ofviable graft and reduced cavitation) tissue and reduced cavitation) 1/5subject—signal intensity 1/5 subject—signal intensity similar to CSF(consistent similar to CSF (consistent with no viable graft tissue) withno viable graft tissue) Year 1 4/5—hyperintense 4/5—hyperintense1/5—signal intensity similar 1/5—signal intensity similar to CSF to CSFYear 2 4/5—hyperintense 4/5—hyperintense 1/5—signal intensity similar1/5—signal intensity similar to CSF to CSF Year 3 4/5—hyperintense4/5—hyperintense 1/5—signal intensity similar 1/5—signal intensitysimilar to CSF to CSF Year 4 4/5—hyperintense 4/5—hyperintense1/5—signal intensity similar 1/5—signal intensity similar to CSF to CSF

Immunological assessment of AST-OPC1 graft rejection. The one yearprimary protocol included collection of cerebrospinal fluid (CSF) andperipheral blood to evaluate whether the subjects mounted an adaptiveimmune response specific to AST-OPC1 and to monitor for evidence of CNSinflammation at the time of tacrolimus discontinuation. CSF wascollected via lumbar puncture immediately prior to surgery for AST-OPC1injection and at Day 60. Peripheral blood was collected at baseline andat frequent intervals through one year follow-up. Additional bloodsamples were collected under the long-term follow-up protocol.

The immune monitoring assays were designated as exploratory assessmentsunder the one year primary protocol and the long-term follow-upprotocol. Serum and CSF from the subjects were assessed for the possibledevelopment of antibodies to the specific human leukocyte antigens (HLA)on AST-OPC1 using the FlowPRA® assay (One Lamba, Inc.). Potentialdevelopment of a cellular immune response to AST-OPC1 was assessed usinga mixed lymphocyte reaction (MLR) assay using peripheral blood monocytes(PBMC) from the subject and AST-OPC1. The MLR was detected using theenzyme-linked immunosorbent spot (ELISPOT) assay for lymphocyteproduction of interferon-gamma (IFN-γ).

Study results revealed that there were no observed increases in T cellmediated responses against AST-OPC1 that were sustained at successivetime points post-transplant for any of the 5 subjects tested (FIG. 13).There were no sustained increases in serum HLA-DSA (donor specificantibodies) from all 5 subjects tested.

In conclusion, based on the results from the immune monitoring assays,there was no detection of humoral or cellular immune responses againstAST-OPC1 that were sustained at successive time points through 1 year offollow-up.

Efficacy results. No subjects exhibited evidence of major sensoryneurological improvement or deterioration through year 2 of follow-up.(FIG. 14). Given the low dosage of AST-OPC1 cells administered in thefirst clinical study, lack of detectable improvement in sensoryneurological function was expected.

Example 9: Quantification of Parenchymal Cavitation at theContusion/Injury Site in Cervical Spinal Cord Injury

Contusion site parenchymal cavitation was assessed and quantitated inrats with cervical spinal cord injury.

Athymic nude rats [Crl:NIH-Foxn lrnu] received a 150 kdyne contusioninjury at C5 using the Infinite Horizons Impactor (Precision Systems &Instrumentation, Fairfax, Va.) and were implanted with 2×10⁶ AST-OPC1cells or HBSS vehicle control at 6-9 days post-injury. Nine monthspost-implantation, the lesion sites were examined histologically and theresults were quantitated.

To examine the anatomical effects of AST-OPC1 transplantation onprogression of the secondary injury contusion cavity, spinal cord tissuewas cut on the longitudinal (horizontal) plane to allow visualization ofthe rostrocaudal extent of the injury site and the grafted AST-OPC1cells.

Contusion site cavitation measurements were performed blind with respectto treatment group on horizontal sections of spinal cord processed forHematoxylin and Eosin staining. Stained sections encompassing the injuryarea and graft were imaged at 2.5× magnification using a Zeiss Axioskop2 Plus microscope (Carl Zeiss, Gottingen, Germany). One representativespinal cord tissue section near the level of the central canal from eachanimal was chosen for measurements of cavitation area. Measurements wereperformed using Image J software (U.S. National Institutes of Health,Bethesda, Md.) to manually trace the perimeter of each area ofcavitation within the contusion injury site. The initial measurementoutput in pixels was converted to millimeters, such that the totalcavitation area for each animal was expressed in units of squaremillimeters. Scatter plots of individual cavitation area measurements,as well as the mean cavitation area and standard error of the mean (SEM)was calculated for each treatment group and displayed graphically usingJMP software (V 11.0.0, SAS Institute, Inc., Cary, N.C.).

To identify myelinated fibers with the spinal cord, tissues were stainedwith Eriochrome (Solochrome) cyanine solution, followed bydifferentiation in 10% iron alum. Slides were counterstained withRuben's Eosin-Phloxine (Biocare Medical, Concord, Calif.), dehydratedthrough a sequential ethanol series, cleared with xylenes andcoverslipped with EcoMount (Biocare Medical, Concord, Calif.).

Consistent with our previous studies in which spinal cord contusion andAST-OPC1 transplantation occurred at the thoracic level, the parenchymalcavitation that is expected secondary to a contusion injury was notobserved in majority of animals that received AST-OPC1 (compare examplesof cavitation in HBSS-treated animals, FIG. 15B, versus reducedcavitation in AST-OPC1-treated animals, FIG. 15A). While cavityformation was not prevented in all animals that received AST-OPC1,parenchymal cavitation was not observed in 124 of 129 animals thatreceived AST-OPC1 and were assessed for micropathology. Of the 62animals that received AST-OPC1 Lot A, two animals had measureable areasof injury-induced parenchymal cavitation (3%), and of the 67 animalsthat received AST-OPC1 Lot B and were assessed for micropathology, threeanimals had measureable areas of injury-induced parenchymal cavitation(4%). In contrast, 7 of 12 animals that received HBSS vehicle hadmeasurable parenchymal cavitation at the injury site (58%). In addition,of the animals with measureable cavitation at the injury site, theaverage cavitation area was reduced in animals treated with AST-OPC1relative to those treated with HBSS (mean cavitation area±standarddeviation: AS T-OPC1=0.64±0.88 mm²; HBSS=1.33±0.91 mm²). The overallmean cavitation areas are shown for each treatment group in FIG. 15C.

What is claimed is:
 1. A method of reducing spinal cord injury-inducedparenchymal cavitation in a human subject with an acute spinal cordinjury, the method comprising directly injecting into the spinal cordinjury site of said subject a composition comprising human pluripotentstem cell-derived oligodendrocyte progenitor cells (OPCs) that arecapable of engrafting at a spinal cord injury site.
 2. The methodaccording to claim 1, wherein administering the composition comprisesdirectly injecting the composition into the spinal cord injury siteapproximately 5 mm caudal of the spinal cord injury epicenter.
 3. Themethod according to claim 1, further comprising administering to thesubject a low dose immunosuppressant regimen.
 4. The method according toclaim 3, wherein the immunosuppressant regimen comprises a tacrolimusdose of about 0.03 mg/kg/day per os, adjusted to maintain a trough bloodconcentration of about 3-7 ng/mL through about day 46 following theadministering of the composition comprising a population of humanpluripotent stem cell-derived OPCs, followed by tapering off anddiscontinuing the immunosuppressant regimen at about day 60 followingthe administering of the composition.
 5. The method according to claim1, wherein the OPCs are capable of remaining within the spinal cordinjury site of said subject for a period of about 180 days or longerfollowing the administration of the composition to the spinal cordinjury site.
 6. The method according to claim 1, wherein the OPCs arecapable of remaining within the spinal cord injury site of said subjectfor a period of about 2 years or longer following the administration ofthe composition to the spinal cord injury site.
 7. The method accordingto claim 1, wherein the OPCs are capable of forming a tissue matrix inthe spinal cord injury site of said subject within about 180 days orless, thereby reducing spinal cord injury-induced parenchymalcavitation.
 8. The method according to claim 1, wherein the subject hasa thoracic or cervical spinal cord injury.
 9. The method according toclaim 1, wherein the composition comprises between about 2×10⁶ and about50×10⁶ cells.
 10. A method of inducing myelin repair or remyelination ina subject comprising administering a therapeutically effective amount ofa composition comprising a population of human pluripotent stemcell-derived oligodendrocyte progenitor cells (OPCs) capable ofsecreting MCP-1 and one or more factors selected from Clusterin, ApoE,TIMP1 and TIMP2; wherein the OPCs are capable of reducing spinal cordinjury-induced parenchymal cavitation in a human subject when injectedinto the spinal cord injury site.
 11. The method of claim 10, whereinthe OPCs are injected into the spinal cord injury site approximately 5mm caudal of the spinal cord injury epicenter of said subject.
 12. Themethod of claim 10, wherein said OPCs are capable of engrafting at saidspinal cord injury site of said subject following implantation of a doseof the composition into said spinal cord injury site; and remainingwithin said spinal cord injury site for a period of 180 days or longer.13. The method according to claim 10, further comprising treating thesubject to reduce immune rejection of the OPCs.
 14. The method accordingto claim 10, further comprising administering an anti-inflammatory agentto the subject.
 15. The method according to claim 10, wherein thesubject has been diagnosed with a disease or pathology selected from:multiple sclerosis, leukodystrophy, Guillain-Barre Syndrome,Charcot-Marie-Tooth neuropathy, Tay-Sachs disease, Niemann-Pick disease,Gaucher disease, and Hurler Syndrome.
 16. The method according to claim10, wherein the subject has been diagnosed with a condition selectedfrom inflammation, stroke, immune disorders, metabolic disorders, andnutritional deficiencies.
 17. The method according to claim 10, whereinthe subject has suffered a traumatic injury resulting in a loss ofmyelination.
 18. The method according to claim 17, wherein the traumaticinjury is an acute spinal cord injury.
 19. The method according to claim17, wherein the traumatic injury is cervical spinal cord injury.
 20. Themethod according to claim 17, wherein the traumatic injury is thoracicspinal cord injury.